Prolonged delivery of peptides

ABSTRACT

There are disclosed methods for the treatment of non-insulin dependent diabetes mellitus in a mammal comprising the prolonged administration of GLP-1 (7-37), and related peptides. Also disclosed are compositions to prolong the administration of the peptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/943,084, filed Aug. 31, 2001, now U.S. Pat. No. 6,828,303, which is acontinuation of U.S. patent application Ser. No. 08/472,349, filed Jun.7, 1995, now U.S. Pat. No. 6,284,727 B1, which is a continuation of U.S.patent application Ser. No. 08/181,655, filed Jan. 25, 1994, nowabandoned, which is a continuation-in-part of U.S. patent applicationSer. No. 08/044,133, filed Apr. 7, 1993, now abandoned. The contents ofthese documents are incorporated herein by reference in their entirety.

BACKGROUND ART

The present invention relates to compositions and methods for thetreatment of Diabetes Mellitus. More specifically, the present inventionrelates to compositions to prolong the administration of glucagon-likepeptide 1 (GLP-1), and derivatives thereof. These compositions areuseful in treatment of Non-Insulin Dependent Diabetes Mellitus (NIDDM).

The amino acid sequence of GLP-1 is known as:His-Asp-Glu-Phe-Glu-Arg-His-Ala- (SEQUENCE ID NO:1)Glu-Gly-Thr-The-Thr-Ser-Asp-Val- Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu- Val-Lys-Gly-Arg-Gly

GLP-1 is disclosed by Lopez, L. C., et al., P.N.A.S., USA 80: 5485-5489(1983); Bell, G. I., et al., Nature 302: 716-718 (1983); Heinrich, G. etal., Endocrinol. 115: 2176-2181 (1984) and Ghiglione, M., et al.,Diabetologia 27: 599-600 (1984).

During processing in the pancreas and intestine, GLP-1 is converted to a31 amino acid peptide having amino acids 7-37 of GLP-1, hereinafter thispeptide is referred to as GLP-1 (7-37).

This peptide has been shown to have insulinotropic activity, that is, itis able to stimulate, or cause to be stimulated, the synthesis orexpression of the hormone insulin. Because of this insulinotropicactivity, GLP-1 (7-37) is alternatively referred to as insulinotropin.

GLP-1 (7-37) has the following amino acid sequence:His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:2)Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arq-Gly.

GLP-1 (7-37), certain derivatives thereof and the use thereof to treatDiabetes mellitus in a mammal are disclosed in U.S. Pat. Nos. 5,118,666('666 patent) and 5,120,712 ('712 patent), the disclosures of thesepatents being incorporated herein by reference. The derivatives of GLP-1(7-37) disclosed in the '666 and '712 patents include polypeptides whichcontain or lack one of more amino acids that may not be present in thenaturally occurring sequence. Further derivatives of GLP-1 (7-37)disclosed in the '666 and '712 patents include certain C-terminal salts,esters and amides where the salts and esters are defined as OM where Mis a pharmaceutically acceptable cation or a lower (C₁-C₆) branched orunbranched alkyl group and the amides are defined as —NR², R³ where R²and R³ are the same or different and are selected from the groupconsisting of hydrogen and a lower (C₁-C₆) branched or unbranched alkylgroup.

Certain other polypeptides, alternatively referred to as truncated GLP-1or truncated insulinotropin, having insulinotropic activity and thederivatives thereof are disclosed in PCT/US 89/01121 (WO 90/11296).Those polypeptides, referred to therein as GLP-1 (7-36), GLP-1 (7-35)and GLP-1 (7-34) have the following amino acid sequences, respectively.His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:3)Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg; His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE IDNO:4) Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly; and His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE IDNO:5) Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys;

Derivatives of the polypeptides disclosed in PCT/US89/01121 includepolypeptides having inconsequential amino acid substitutions, oradditional amino acids to enhance coupling to carrier protein or toenhance the insulinotropic effect thereof. Further derivatives ofinsulinotropin disclosed in PCT/US89/01121 include certain C-terminalsalts, esters and amides where the salts and esters are defined as OMwhere M is a pharmaceutically acceptable cation or a lower branched orunbranched alkyl group and the amides are defined as —NR², R³ where R²and R³ are the same or different and are selected from the groupconsisting of hydrogen and a lower branched or unbranched alkyl group.

DISCLOSURE OF THE INVENTION

In one embodiment, the present invention is directed to a method for thetreatment of non-insulin dependent diabetes mellitus in a mammal in needof such treatment comprising the repeated administration over anextended period of time of a compound with prolonged action after eachadministration, said prolonged action necessary to achieve sustainedglycemic control in such mammals, said compound selected from the groupconsisting of:

(a) a peptide having the amino acid sequence:His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:2)Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly;

(b) a peptide having the amino acid sequence:His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:7)Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-X

wherein X is selected from the group consisting of: (A) Lys, (B)Lys-Gly, and (C) Lys-Gly-Arg;

-   -   (c) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein W is an amino acid sequence selected from the group consistingof His-Asp-Glu-Phe-Glu-Arg-His-Ala- (SEQUENCE ID NO:1)Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val- Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu- Val-Lys-Gly-Arg-Gly andHis-Asp-Glu-Phe-Glu-Arg-His-Ala- (SEQUENCE ID NO:6)Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val- Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu- Val-Lys-Gly-Arg

-   -   which derivative when processed in a mammal results in a        polypeptide derivative having an insulinotropic activity;    -   (d) a derivative of a polypeptide comprising the primary        structure        H₂N—R—COOH

wherein R is an amino acid sequence selected from the group consistingof His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:2)Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly; His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCEID NO:3) Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly-Arg;His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:4)Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly; and His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE IDNO:5) Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys;

-   -   and    -   (e) a derivative of said peptides (a) through (d) wherein said        derivative is selected from the group consisting of:    -   (1) a pharmaceutically acceptable acid addition salt of said        peptides;    -   (2) a pharmaceutically acceptable carboxylate salt of said        peptides;    -   (3) a pharmaceutically acceptable alkali addition salt of said        peptides;    -   (4) a pharmaceutically acceptable lower alkyl ester of said        peptides; and    -   (5) a pharmaceutically acceptable amide of said peptides wherein        said pharmaceutically acceptable amide is selected from the        group consisting of amide, lower alkyl amide and lower dialkyl        amide.

Preferred is the method wherein said administration is subcutaneous.

Also preferred is the method wherein said administration isintramuscular.

Also preferred is the method wherein said administration is transdermal.

Also especially preferred is the method wherein said administration isby an infusion pump.

Also preferred is the method wherein said administration is by oralinhalation.

Also preferred is the method wherein said administration is by nasalinhalation.

Also preferred is the method wherein said administration isgastrointestinal.

In another embodiment, the present invention is directed to acomposition of matter comprising;

-   -   (i) a compound selected from the group consisting of:

(a) a peptide having the amino acid sequence:His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:2)Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly;

(b) a peptide having the amino acid sequence:His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- (SEQUENCE ID NO:7)Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-AlaTrp-Leu-Val-X

wherein X is selected from the group consisting of: (A) Lys, (B)Lys-Gly, and (C) Lys-Gly-Arg;

-   -   (c) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein W is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:1) His-Asp-Glue-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- Gly and (SEQUENCE IDNO:6) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg

-   -   which derivative when processed in a mammal results in a        polypeptide derivative having an insulinotropic activity;    -   (d) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein R is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:2) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQUENCE ID NO:3)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly-Arg(SEQUENCE ID NO:4) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly and(SEQUENCE ID NO:5) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys;

-   -   a derivative of said peptides (a) through (d) wherein said        derivative is selected from the group consisting of:    -   (1) a pharmaceutically acceptable acid addition salt of said        peptides;    -   (2) a pharmaceutically acceptable carboxylate salt of said        peptides;    -   (3) a pharmaceutically acceptable alkali addition salt of said        peptides;    -   (4) a pharmaceutically acceptable lower alkyl ester of said        peptides; and    -   (5) a pharmaceutically acceptable amide of said peptides wherein        said pharmaceutically acceptable amide is selected from the        group consisting of amide, lower alkyl amide and lower dialkyl        amide, and    -   (ii) a polymer capable of prolonging the action of said compound        to achieve sustained glycemic control.

Especially preferred is the composition wherein said polymer is a lowmolecular weight polymer.

Further especially preferred is a composition wherein said polymer isselected from the group consisting of polyethylene glycol,polyvinylpyrrolidone, polyvinylalcohol, polyoxyethylene-polyoxypropylenecopolymers, polysaccharides selected from the group consisting ofcellulose, cellulose derivatives, chitosan, acacia gum, karaya gum, guargum, xanthan gum, tragacanth, alginic acid, carrageenan, agarose, andfurcellarans, dextran, starch, starch derivatives, hyaluronic acid,polyesters, polyamides, polyanhydrides, and polyortho esters, withespecially preferred polymers selected from the group consisting ofpolyethylene glycol and polyvinylpyrrolidone.

In another embodiment, the present invention is directed to acomposition of matter comprising;

-   -   (i) a compound selected from the group consisting of:

(a) a peptide having the amino acid sequence: (SEQUENCE ID NO:2)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly;

(b) a peptide having the amino acid sequence: (SEQUENCE ID NO:7)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-X

wherein X is selected from the group consisting of: (A) Lys, (B)Lys-Gly, and (C) Lys-Gly-Arg;

-   -   (c) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein W is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:1) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- Gly and (SEQUENCE IDNO:6) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg

-   -   which derivative when processed in a mammal results in a        polypeptide derivative having an insulinotropic activity;    -   (d) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein R is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:2) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly; (SEQUENCE ID NO:3)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg; (SEQUENCE ID NO:4)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly;and (SEQUENCE ID NO:5) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys;

-   -   and    -   a derivative of said peptides (a) through (d) wherein said        derivative is selected from the group consisting of:    -   (1) a pharmaceutically acceptable acid addition salt of said        peptides;    -   (2) a pharmaceutically acceptable carboxylate salt of said        peptides;    -   (3) a pharmaceutically acceptable alkali addition salt of said        peptides;    -   (4) a pharmaceutically acceptable lower alkyl ester of said        peptides; and    -   (5) a pharmaceutically acceptable amide of said peptides wherein        said pharmaceutically acceptable amide is selected from the        group consisting of amide, lower alkyl amide and lower dialkyl        amide, and    -   (ii) a pharmaceutically acceptable water-immiscible oil        suspension capable of prolonging administration of said        compound.

Especially preferred is the composition wherein said oil is selectedfrom the group consisting of peanut oil, sesame oil, almond oil, castoroil, camellia oil, cotton seed oil, olive oil, corn oil, soy oil,safflower oil, coconut oil, esters of fatty acids, and esters of fattyalcohols. Further especially preferred is the composition furthercomprising a wetting agent, especially a nonionic surfactant.

More further especially preferred is the composition further comprisinga suspending agent.

In another embodiment, the present invention is directed to acomposition of matter comprising;

-   -   (i) a compound selected from the group consisting of:

(a) a peptide having the amino acid sequence: (SEQUENCE ID NO:2)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly;

(b) a peptide having the amino acid sequence: (SEQUENCE ID NO:7)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-X

wherein X is selected from the group consisting of: (A) Lys, (B)Lys-Gly, and (C) Lys-Gly-Arg;

-   -   (c) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein W is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:1) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- Gly and (SEQUENCE IDNO:6) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg

-   -   which derivative when processed in a mammal results in a        polypeptide derivative having an insulinotropic activity;    -   (d) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein R is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:2) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQUENCE ID NO:3)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly-Arg(SEQUENCE ID NO:4) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly and(SEQUENCE ID NO:5); His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys;

-   -   and    -   a derivative of said peptides (a) through (d) wherein said        derivative is selected from the group consisting of:    -   (1) a pharmaceutically acceptable acid addition salt of said        peptides;    -   (2) a pharmaceutically acceptable carboxylate salt of said        peptides;    -   (3) a pharmaceutically acceptable alkali addition salt of said        peptides;    -   (4) a pharmaceutically acceptable lower alkyl ester of said        peptides; and    -   (5) a pharmaceutically acceptable amide of said peptides wherein        said pharmaceutically acceptable amide is selected from the        group consisting of amide, lower alkyl amide and lower dialkyl        amide, and    -   (ii) zinc (II), which is complexed with the peptide.

Preferred is the composition capable of sustained glycemic action.

Especially preferred is the composition wherein the zinc product isamorphous.

Also especially preferred is the composition wherein the zinc product iscrystalline.

In yet another embodiment, the present invention is directed to acomposition of matter comprising;

-   -   (i) a compound selected from the group consisting of:

(a) a peptide having the amino acid sequence: (SEQUENCE ID NO:2)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly;

(b) a peptide having the amino acid sequence: (SEQUENCE ID NO:7)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-X

wherein X is selected from the group consisting of: (A) Lys, (B)Lys-Gly, and (C) Lys-Gly-Arg;

-   -   (c) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein W is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:1) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- Gly and (SEQUENCE IDNO:6) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg

-   -   and which derivative when processed in a mammal results in a        polypeptide derivative having an insulinotropic activity;    -   (d) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein R is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:2) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQUENCE ID NO:3)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly-Arg(SEQUENCE ID NO:4) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly and(SEQUENCE ID NO:5) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys;

-   -   and    -   a derivative of said peptides (a) through (d) wherein said        derivative is selected from the group consisting of:    -   (1) a pharmaceutically acceptable acid addition salt of said        peptides;    -   (2) a pharmaceutically acceptable carboxylate salt of said        peptides;    -   (3) a pharmaceutically acceptable alkali addition salt of said        peptides;    -   (4) a pharmaceutically acceptable lower alkyl ester of said        peptides; and    -   (5) a pharmaceutically acceptable amide of said peptides wherein        said pharmaceutically acceptable amide is selected from the        group consisting of amide, lower alkyl amide and lower dialkyl        amide, and    -   (ii) a metal selected from the group consisting of Ni (II), Co        (II), Mg (II), Ca (II), K (I), Mn (II), Fe(II), and Cu(II).

In yet another embodiment, the present invention is directed to acomposition of matter comprising;

-   -   (i) a compound selected from the group consisting of:

(a) a peptide having the amino acid sequence: (SEQUENCE ID NO:2)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly;

(b) a peptide having the amino acid sequence: (SEQUENCE ID NO:7)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-X

wherein X is selected from the group consisting of: (A) Lys, (B)Lys-Gly, (C) Lys-Gly-Arg;

-   -   (c) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein W is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:1) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- Gly and (SEQUENCE IDNO:6) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg

-   -   which derivative when processed in a mammal results in a        polypeptide derivative having an insulinotropic activity;    -   (d) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein R is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:2) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly; (SEQUENCE ID NO:3)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg; (SEQUENCE ID NO:4)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly;and (SEQUENCE ID NO:5) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Gly-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys;

-   -   and    -   a derivative of said peptides (a) through (d) wherein said        derivative is selected from the group consisting of:    -   (1) a pharmaceutically acceptable acid addition salt of said        peptides;    -   (2) a pharmaceutically acceptable carboxylate salt of said        peptides;    -   (3) a pharmaceutically acceptable alkali addition salt of said        peptides;    -   (4) a pharmaceutically acceptable lower alkyl ester of said        peptides; and    -   (5) a pharmaceutically acceptable amide of said peptides wherein        said pharmaceutically acceptable amide is selected from the        group consisting of amide, lower alkyl amide and lower dialkyl        amide, and    -   (ii) a basic polypeptide, wherein such composition is an aqueous        suspension capable of sustained glycemic control.

Especially preferred is the composition wherein the basic polypeptide isprotamine.

In yet another embodiment, the present invention is directed to acomposition of matter comprising;

-   -   (i) a compound selected from the group consisting of:

(a) a peptide having the amino acid sequence: (SEQUENCE ID NO:2)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly;

(b) a peptide having the amino acid sequence: (SEQUENCE ID NO:7)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-X

wherein X is selected from the group consisting of: (A) Lys, (B)Lys-Gly, (C) Lys-Gly-Arg;

-   -   (c) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein W is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:1) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- Gly and (SEQUENCE IDNO:7) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg

-   -   which derivative when processed in a mammal results in a        polypeptide derivative having an insulinotropic activity;    -   (d) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein R is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:2) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly; (SEQUENCE ID NO:3)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg; (SEQUENCE ID NO:4)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly;and (SEQUENCE ID NO:5) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys;

-   -   and    -   a derivative of said peptides (a) through (d) wherein said        derivative is selected from the group consisting of:    -   (1) a pharmaceutically acceptable acid addition salt of said        peptides;    -   (2) a pharmaceutically acceptable carboxylate salt of said        peptides;    -   (3) a pharmaceutically acceptable alkali addition salt of said        peptides;    -   (4) a pharmaceutically acceptable lower alkyl ester of said        peptides; and    -   (5) a pharmaceutically acceptable amide of said peptides wherein        said pharmaceutically acceptable amide is selected from the        group consisting of amide, lower alkyl amide and lower dialkyl        amide, and    -   (ii) a phenolic compound, wherein such composition is an aqueous        suspension capable of sustained glycemic control.

Especially preferred is the composition wherein said phenolic compoundis selected from the group consisting of phenol, cresol, resorcinol, andmethyl/araben.

In yet another embodiment, the present invention is directed to acomposition of matter comprising;

-   -   (i) a compound selected from the group consisting of:

(a) a peptide having the amino acid sequence: (SEQUENCE ID NO:2)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly;

(b) a peptide having the amino acid sequence: (SEQUENCE ID NO:7)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-X

wherein X is selected from the group consisting of: (A) Lys, (B)Lys-Gly, (C) Lys-Gly-Arg;

-   -   (c) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein W is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:1) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- Gly and (SEQUENCE IDNO:6) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg

-   -   which derivative when processed in a mammal results in a        polypeptide derivative having an insulinotropic activity;    -   (d) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein R is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:2) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly; (SEQUENCE ID NO:3)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg; (SEQUENCE ID NO:4)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly;and (SEQUENCE ID NO:5) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys;

-   -   and    -   a derivative of said peptides (a) through (d) wherein said        derivative is selected from the group consisting of:    -   (1) a pharmaceutically acceptable acid addition salt of said        peptides;    -   (2) a pharmaceutically acceptable carboxylate salt of said        peptides;    -   (3) a pharmaceutically acceptable alkali addition salt of said        peptides;    -   (4) a pharmaceutically acceptable lower alkyl ester of said        peptides; and    -   (5) a pharmaceutically acceptable amide of said peptides wherein        said pharmaceutically acceptable amide is selected from the        group consisting of amide, lower alkyl amide and lower dialkyl        amide, and    -   (ii) a basic polypeptide and a phenolic compound, wherein such        composition is an aqueous suspension capable of sustained        glycemic control.

In another embodiment, the present invention is directed to acomposition of matter comprising;

-   -   (i) a compound selected from the group consisting of:

(a) a peptide having the amino acid sequence: (SEQUENCE ID NO:2)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly;

(b) a peptide having the amino acid sequence: (SEQUENCE ID NO:7)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-X

wherein X is selected from the group consisting of: (A) Lys, (B)Lys-Gly, and (C) Lys-Gly-Arg;

-   -   (c) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein W is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:1) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- Gly and (SEQUENCE IDNO:6) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg

-   -   which derivative when processed in a mammal results in a        polypeptide derivative having an insulinotropic activity;    -   (d) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein R is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:2) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly; (SEQUENCE ID NO:3)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg; (SEQUENCE ID NO:4)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly;and (SEQUENCE ID NO:5) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys;

-   -   and    -   a derivative of said peptides (a) through (d) wherein said        derivative is selected from the group consisting of:    -   (1) a pharmaceutically acceptable acid addition salt of said        peptides;    -   (2) a pharmaceutically acceptable carboxylate salt of said        peptides;    -   (3) a pharmaceutically acceptable alkali addition salt of said        peptides;    -   (4) a pharmaceutically acceptable lower alkyl ester of said        peptides; and    -   (5) a pharmaceutically acceptable amide of said peptides wherein        said pharmaceutically acceptable amide is selected from the        group consisting of amide, lower alkyl amide and lower dialkyl        amide, and    -   (ii) a basic polypeptide, a phenolic compound, and a metal ion        wherein said composition is an aqueous suspension capable of        sustained glycemic control.

Preferred is the composition wherein said basic polypeptide isprotamine.

Also preferred is the composition wherein said metal ion is zinc.

In another embodiment, the present invention is directed to acomposition of matter comprising;

-   -   (i) a compound selected from the group consisting of:

(a) a peptide having the amino acid sequence: (SEQUENCE ID NO:2)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly;

(b) a peptide having the amino acid sequence: (SEQUENCE ID NO:7)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-X

wherein X is selected from the group consisting of: (A) Lys, (B)Lys-Gly, (C) Lys-Gly-Arg;

-   -   (c) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein W is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:1) His-Asp-Glu-Phe-Glu-Arq-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly--Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- Gly and (SEQUENCE IDNO:6) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg

-   -   which derivative when processed in a mammal results in a        polypeptide derivative having an insulinotropic activity;    -   (d) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein R is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:2) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly; (SEQUENCE ID NO:3)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg; (SEQUENCE ID NO:4)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly;and (SEQUENCE ID NO:5) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys;

-   -   and    -   a derivative of said peptides (a) through (d) wherein said        derivative is selected from the group consisting of:    -   (1) a pharmaceutically acceptable acid addition salt of said        peptides;    -   (2) a pharmaceutically acceptable carboxylate salt of said        peptides;    -   (3) a pharmaceutically acceptable alkali addition salt of said        peptides;    -   (4) a pharmaceutically acceptable lower alkyl ester of said        peptides; and    -   (5) a pharmaceutically acceptable amide of said peptides wherein        said pharmaceutically acceptable amide is selected from the        group consisting of amide, lower alkyl amide and lower dialkyl        amide, and    -   (ii) said peptides and derivatives thereof having been subjected        to conditions resulting in amorphous crystalline formation.

Preferred is the composition wherein said conditions are high shear,exposure to salts; or combinations thereof.

Especially preferred is the composition wherein said salt is selectedfrom the group consisting of ammonium sulfate, sodium sulfate, lithiumsulfate, lithium chloride, sodium citrate, ammonium citrate, sodiumphosphate, potassium phosphate, sodium chloride, potassium chloride,ammonium chloride, sodium acetate, ammonium acetate, magnesium sulfate,calcium chloride, ammonium nitrate, and sodium formate; and combinationsthereof.

In still another embodiment, the present invention is directed to acomposition of matter comprising;

-   -   (i) a compound selected from the group consisting of:

(a) a peptide having the amino acid sequence: (SEQUENCE ID NO:2)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly;

(b) a peptide having the amino acid sequence: (SEQUENCE ID NO:7)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-X

wherein X is selected from the group consisting of: (A) Lys, (B)Lys-Gly, (C) Lys-Gly-Arg;

-   -   (c) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein W is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:1) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- Gly and (SEQUENCE IDNO:6) His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys Gly-Arg

-   -   which derivative when processed in a mammal results in a        polypeptide derivative having an insulinotropic activity;    -   (d) a derivative of a polypeptide comprising the primary        structure        H₂N—W—COOH

wherein R is an amino acid sequence selected from the group consistingof (SEQUENCE ID NO:2) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly; (SEQUENCE ID NO:3)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg; (SEQUENCE ID NO:4)His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly;and (SEQUENCE ID NO:5) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Lys;

-   -   and    -   a derivative of said peptides (a) through (d) wherein said        derivative is selected from the group consisting of:    -   (1) a pharmaceutically acceptable acid addition salt of said        peptides;    -   (2) a pharmaceutically acceptable carboxylate salt of said        peptides;    -   (3) a pharmaceutically acceptable alkali addition salt of said        peptides;    -   (4) a pharmaceutically acceptable lower alkyl ester of said        peptides; and    -   (5) a pharmaceutically acceptable amide of said peptides wherein        said pharmaceutically acceptable amide is selected from the        group consisting of amide, lower alkyl amide and lower dialkyl        amide, and    -   (ii) a liposome delivery system.

Especially preferred is the composition wherein said liposome isphospholipid based.

Also especially preferred is the composition wherein said liposome isnon-phospholipid based.

The present invention is also directed to the treatment of non-insulindependent diabetes mellitus in a mammal in need of such treatmentcomprising the prolonged administration of the compositions of thepresent invention. *

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of a prolonged infusion (7 hours) of 4 ng/kg/mininsulinotropin on plasma glucose levels in patients with NIDDM.

FIG. 2 shows the effect of a short infusion (60 minutes) of 10 ng/kg/mininsulinotropin on plasma glucose levels in patients with NIDDM.

FIG. 3 shows the effect of a prolonged infusion (7 hours) of 2 ng/kg/minand 4 ng/kg/min of insulinotropin on plasma glucose levels in patientswith NIDDM.

FIG. 4. Mean (n=3) Plasma Concentration of Insulinotropin in Rats AfterSubcutaneous Administration of Single 0.5 mg/0.5 ml Doses in DifferentAqueous Suspensions (AS).

FIG. 5. Mean (n=3) Plasma Concentration of Insulinotropin in Rats AfterSubcutaneous Administration of Single 0.5 mg/0.5 ml Doses in DifferentAqueous Suspensions (AS).

FIG. 6. Mean (n=3) Plasma Concentration of Insulinotropin in Rats AfterSubcutaneous Administration of Single 0.5 mg/0.5 ml Doses in DifferentAqueous Suspensions (AS).

FIG. 7. Mean (n=3) Plasma Concentration of Insulinotropin in Rats AfterSubcutaneous Administration of Single 0.5 mg/0.5 ml Doses in DifferentAqueous Suspensions (AS).

FIG. 8. Mean (n=3) Plasma Concentration of Insulinotropin in Rats AfterSubcutaneous Administration of Single 0.5 mg/0.13 ml Doses in DifferentAqueous Suspensions (AS).

FIG. 9. Mean (n=3) Plasma Concentration of Insulinotropin in Rats AfterSubcutaneous Administration of Single 0.5 mg/0.13 ml Doses in DifferentAqueous Suspensions (AS).

FIG. 10 shows pharmacokinetic studies of an insulinotropin zincprecipitate.

MODES OF CARRYING OUT THE INVENTION

Unless otherwise indicated, the term “derivative”, as used throughoutthis Specification and the appendant claims, includes, but is notlimited to, polypeptides comprising the primary structure shown, whereinone or more L-amino acids are included at the C-terminus thereof;wherein the C-terminal carboxyl group forms an ester with a (C₁-C₆)straight or branched chain alkyl group; wherein the C-terminal carboxylgroup forms a carboxamide or substituted carboxamide; wherein the acidicamino acid residues (Asp and/or Glu) form an ester or carboxamide; andcombinations thereof.

Included within the scope of this invention are polypeptides havinghomology to the peptides described above, which homology is sufficientto impart insulinotropic activity to such polypeptides. Also includedwithin the scope of this invention are variants of the polypeptidesdescribed above, which variants comprise inconsequential amino acidsubstitutions and have insulinotropic activity.

Glucagon-like Peptide-1 (7-37), its isolation, characterization, and useto treat Diabetes mellitus are disclosed in U.S. Pat. Nos. 5,118,666 and5,120,712, the disclosures of these patents in their entirety beingincorporated herein by reference.

In the present invention, it has now been discovered that prolongedplasma elevations of GLP-1, and related polypeptides, are necessaryduring the meal arid beyond to achieve sustained glycemic control inpatients with Non Insulin Dependent Diabetes Mellitus. It hassurprisingly been found that raising GLP-1, and related peptides, aroundmeal time alone, even for periods of up to one hour, will not adequatelycontrol the glucose levels. Thus, administration of GLP-1, and relatedpeptides, requires a prolonged delivery system. This prolonged deliverysystem leads to an enhancing of insulin action.

The phrase “enhancing insulin action”, as used throughout thisSpecification and the appendant claims, includes, but is not limited to,one or more of increasing insulin synthesis, increasing insulinsecretion, increasing glucose uptake by muscle and fat and decreasingglucose production by the liver.

The polypeptides of this invention are prepared by various methods wellknown to those skilled in the art. For example, the polypeptides can besynthesized using automated peptide synthesizers such as an AppliedBiosystems (ABI) 430A solid phase peptide synthesizer. Alternatively,the polypeptides of this invention can be prepared using recombinant DNAtechnology wherein a DNA sequence coding for the polypeptide is operablylinked to an expression vector and used to transform an appropriate hostcell. The transformed host cell is then cultured under conditionswhereby the polypeptide will be expressed. The polypeptide is thenrecovered from the culture. Further still, a combination of synthesisand recombinant DNA techniques can be employed to produce the amide andester derivatives of this invention and/or to produce fragments of thedesired polypeptide which are then joined by methods well known to thoseskilled in the art.

Derivatives of the polypeptides according to this invention are preparedby methods well known to those skilled in the art. For example,C-terminal alkyl ester derivatives of the polypeptides of this inventionare prepared by reacting the desired (C₁-C₆) alkanol with the desiredpolypeptide in the presence of a catalytic acid such as HCl. Appropriatereaction conditions for such alkyl ester formation include a reactiontemperature of about 50° C. and reaction times of about 1 hour to about3 hours. Similarly, derivatives of the polypeptides of this inventioncomprising (C₁-C₆) alkyl esters of the Asp and/or Glu residues withinthe polypeptide can be so formed. °

Carboxamide derivatives of the polypeptides of this invention are alsoprepared by solid phase peptide synthesis methods well known to thoseskilled in the art. For example, see, Solid Phase Peptide Synthesis,Stewart, J. M. et al., Pierce Chem. Co. Press, 1984.

Alternatively, or in combination with the above, derivatives of thepolypeptides of this invention can be prepared by modifying the DNAcoding sequence for such polypeptide so that a basic amino acid residueis replaced with a different basic amino acid residue or with an acidacidic or neutral amino acid residue, or an acidic amino acid residue isreplaced with a different acidic amino acid residue or with a basic orneutral amino acid residue, or a neutral amino acid residue is replacedwith a different neutral amino acid residue or with an acidic or basicamino acid residue. Such changes in polypeptide primary sequence canalso be accomplished by direct synthesis of the derivative. Such methodsare well known to those skilled in the art. Of course, such derivatives,to be useful in the practice of this invention, must achieve aninsulinotropic effect.

The insulinotropic activity of a polypeptide derivative according tothis invention is determined as follows.

Pancreatic islets are isolated from pancreatic tissue from normal ratsby a modification of the method of Lacy, P. E., et al., Diabetes,16:35-39 (1967) in which the collagenase digest of pancreatic tissue isseparated on a Ficoll gradient (27%, 23%, 20.5% and 11% in Hanks'balanced salt solution, pH 7.4). The islets are collected from the20.5%/11% interface, washed and handpicked free of exocrine and othertissue under a stereomicroscope. The islets are incubated overnight inRPMI 1640 medium supplemented with 10% fetal bovine serum and containing11 mM glucose at 37° C. and 95% air/5% CO₂. The islets are thentransferred to RPMI 1640 medium supplemented with 10% fetal bovine serumand containing 5.6 mM glucose. The islets are incubated for 60 minutesat 37° C., 95% air/5% CO₂. The polypeptide derivative to be studied isprepared at 1 nM and 10 nM concentrations in RPMI medium containing 10%fetal bovine serum and 16.7 mM glucose. About 8 to 10 isolated isletsare then transferred by pipette to a total volume of 250 μl of thepolypeptide derivative containing medium in 96 well microtiter dishes.The islets are incubated in the presence of the polypeptide derivativeat 37° C., 95% air/5% CO₂ for 90 minutes. Then, aliquots of islet-freemedium are collected and 100 μl thereof are assayed for the amount ofinsulin present by radioimmunoassay using an Equate Insulin RIA Kit(Binax, Inc., Portland, Me.).

Dosages effective in treatment of adult onset diabetes will range fromabout 1 pg/kg to 1,000 μg/kg per day when a polypeptide derivative ofthis invention is administered, for example, intravenously,intramuscularly or subcutaneously. A preferred dosage range forintravenous infusion during and between meals is about 4 to 10 ng/kg/minor about 0.6 to 1.4 μg/day based on a 100 kg patient. It is to beappreciated, however, that dosages outside of that range are possibleand are also within the scope of this invention. The appropriate dosagecan and will be determined by the prescribing physician and will be aresult of the severity of the condition being treated as well as theresponse achieved with the derivative being administered and the age,weight, sex and medical history of the patient.

The prolonged administration may be achieved by subcutaneous,intramuscular, or transdermal means, oral inhalation, nasal inhalation,gastrointestinal, or by means of an infusion pump.

Prolonged administration of GLP-1, and related peptides, may also beachieved by formulation as a solution in various water-soluble polymers.These polymers are generally low molecular weight (<15 kDa) polymers.Non-limiting examples of such low molecular weight polymers includepolyethylene glycol, polyvinylpyrrolidone, polyvinylalcohol andpolyoxyethylene-polyoxypropylene copolymers. Higher molecular weightpolymers may be used. Non-limiting examples of higher molecular weightpolymers include polysaccharides such as cellulose and its derivatives,chitosan, acacia gum, karaya gum, guar gum, xanthan gum, tragacanth,alginic acid, carrageenan, agarose, furcelleran. In the later case,polymers which are degraded in vivo either enzymatically or byhydrolysis are preferred, for example, dextran; starch and itsderivatives, hyaluronic acid, polyesters, polyamides, polyanhydrides andpolyortho esters. The tissue accumulation associated with high molecularweight, non-biodegradable polymers is avoided by using low molecularweight polymers or biodegradable polymers. The formulations typicallycontain GLP-1, or related peptides, at approximately 1 mg/ml, withconcentration dependent on the polymer, but typically at concentrationsup to that which will attain a 50 cps viscosity, and possibly a suitablebuffer, tonicity agent, and preservative. In vivo data in rats and mandemonstrate that the formulations are capable of achieving measurableblood insulinotropin, for example, levels for up to 24 hours. Incontrast, insulinotropin, for example, formulated in phosphate-bufferedsaline results in rapid (˜15 minutes) peak plasma levels, with plasmalevel dropping below detection limits in just over 4 hours. Plasmaconcentration versus time plots suggest that insulinotropin absorptionrate, for example, from the injection site has been significantlyreduced in the presence of the polymers.

GLP-1, and related peptides, may also be formulated as particlessuspended in a pharmaceutically acceptable oil. The preferred oils aretriglycerides. Non-limiting examples of such oils include peanut oil,sesame oil, almond oil, castor oil, camellia oil, cotton seed oil, oliveoil, corn oil, soy oil, safflower oil, and coconut oil. Oils of otherclasses are acceptable, for example, esters of fatty acids and esters offatty alcohols, as long as the oil is immiscible with water and is apoor solvent for the peptide. The formulation may also containappropriate preservatives, wetting agents, and suspending agents. Theweight percent of insulinotropin, for example, in the formulation mayvary from 0.01 to 10%. In vivo data in rats demonstrate that theseformulations are capable of achieving measurable insulinotropin bloodlevels, for example, for up to 24 hours. In contrast, insulinotropin,for example, formulated in phosphate-buffered saline results in rapid(.about. 15 minutes) peak plasma levels, with plasma level droppingbelow detection limits in just over 4 hours. Plasma concentration versustime plots suggest that insulinotropin absorption rate from theinjection site have been significantly reduced in the oil suspensions.

GLP-1, and related peptides, may also be formulated as a low solubilityform for administration by combination with a metal ion, preferably inthe form of a salt. A preferred ion is zinc (II). The combination mayresult in a composition which is amorphous or crystalline. Other metalions may also be used including Ni(II), Co(II), Mg(II), Ca(II), K(I),Mn(II), Fe(II) and Cu(II).

Other forms of prolonged administration include liposomes, eithermultilamellar or unilamellar, the preparation of which is well known tothose skilled in the art. The liposomes, whether multilamellar orunilamellar, may be phospholipid or non-phospholipid based.

Another type of prolonged delivery formulation is an aqueous suspensionof insulinotropin precipitates or aggregates formed by usingprecipitants for example, phenolic compounds or basic polypeptides ormetal ions or salts, and/or by using high shear. More than oneprecipitant can be used at one time. The precipitates can be eithercrystalline or amorphous.

Insulinotropin crystals can be obtained from a solution of the drug inwater by using pH gradient (either high to low or low to high) and/ortemperature gradient and/or salts to reduce solubility. The saltsinclude ammonium citrate, sodium or potassium phosphate, sodium orpotassium or ammonium chloride, sodium or ammonium acetate, magnesiumsulfate, calcium chloride, ammonium nitrate, sodium formate, and anyother salts which can reduce the solubility of the drug. If the saltused for crystallization is not pharmaceutically acceptable, the motherliquor can be substituted by pharmaceutically acceptable medium aftercrystallization is completed. If further reduction of drug solubility isnecessary to achieve a desirable pharmacokinetic profile, the crystalscan be treated by metal ions such as zinc or calcium and/or phenoliccompounds. The treatment can be done by simply incorporating thoseadditives to the crystal suspension.

The solubility of the insulinotropin precipitates or aggregates canrange from less than 1 μg/mL to 500 μg/mL under physiologicalconditions. In vivo data in rats demonstrate that the formulations arecapable of achieving measurable insulinotropin blood levels, forexample, for at least 30 hours.

Aqueous media used for the above formulations can be any kind of buffersystem which can be used for injection or even with pure water. The pHof the final formulation can be any value as long as the formulation isinjectable. Protamine can be added as any kind of salt form (e.g.sulfate, chloride, etc.) or protamine base. Exemplary concentrationranges of the components which can be used for the formulationpreparation are as follows: phenol (0.5 to 5.0 mg/ml), m-cresol (0.5 to5.5 mg/ml), protamine (0.02 to 1.0 mg/ml), zinc (0.10 to 6zinc/insulinotropin molar ratio), sodium chloride (up to 100 mg/ml), andphosphate buffer (5-500 mM).

Other phenolic on non phenolic compounds may also be used. Non-limitingexamples of such compounds include resorcinol, methylparaben,propylparaben, benzyl alcohol, chlorocresol, cresol, benzaldehyde,catecol, pyrogallol, hydroquinone, n-propyl gallate, butylatedhydroxyanisole, butylated hydroxytoluene. Non-limiting examples of basicpolypeptides are polylysine, polyarginine, etc.

Having described the invention in general terms, reference is now madeto specific examples. It is to be understood that these examples are notmeant to limit the present invention, the scope of which is determinedby the appended claims.

EXAMPLE 1 Insulinotropin (1 mg/ml) Suspension Solution A1 Preparation

10 mg of insulinotropin was weighed into a 5 ml volumetric flask.Approximately 4 ml of phosphate buffered saline (PBS) was added to theflask to disperse and dissolve the drug. Sufficient PBS (q.s. amount)was added to fill the flask. 20 mg of insulinotropin was weighed into a10 ml volumetric flask. Approximately 8 ml of PBS was added to the flaskto disperse and dissolve the drug. The q.s. amount of PBS was added tothe flask. The volumes in both flasks were combined by filtering them bya glass syringe through a 0.22μ filter (low protein binding) into a 10ml glass vial. Solution A1 contained insulinotropin 2 mg/ml in PBS.

Solution B1 preparation 8 mg of protamine sulfate and 44 mg of phenolwere weighed into a 10 ml volumetric flask. The q.s. amount of PBS wasadded to dissolve the protamine sulfate and the phenol. This solutionwas filtered through a 0.22μ filter (low protein binding) into a 10 mlglass vial. Solution B1 contained protamine base 0.6 mg/ml and phenol4.4 mg/ml in PBS.

Aqueous Suspension 1

1.5 ml of solution A1 was pipetted into a 3.5 ml type I glass vial. Thecontents of the vial were stirred magnetically while 1.5 ml of solutionB1 was pipetted into the vial. The vial was stoppered and sealed with analuminum shell. The vial contents were stirred gently for 16 hours toallow suspension formation. Aqueous Suspension 1 containedinsulinotropin 1 mg/ml, protamine base 0.3 mg/ml and phenol 2.2 mg/ml inPBS. This suspension was used for in vivo pharmacokinetic studies inrats.

EXAMPLE 2 Insulinotropin (1 mg/ml) Suspension Solution A2 Preparation

10 mg of insulinotropin was weighed into a 5 ml volumetric flask.Approximately 4 ml of PBS was added to the flask to disperse anddissolve the drug. The q.s. amount of PBS was added to the flask. 20 mgof insulinotropin was weighed into a 10 ml volumetric flask.Approximately 8 ml of PBS was added to the flask to disperse anddissolve the drug. The q.s. amount of the PBS was added to the flask.The volumes in both flasks were combined by filtering them by a glasssyringe through a 0.22μ filter into a 10 ml glass vial. Solution A2contained insulinotropin 2 mg/ml in PBS.

Solution B2 Preparation

2 mg of protamine sulfate and 44 mg of phenol were weighed into a 10 mlvolumetric flask. The q.s. amount of PBS was added to the flask todissolve the protamine sulfate and phenol. This solution was filteredthrough a 0.22μ filter into a 10 ml glass vial. Solution B2 containedprotamine base 0.15 mg/ml and phenol 4.4 mg/ml in PBS.

Aqueous Suspension 2

1.5 ml of solution A2 was pipetted into a 3.5 ml type I glass vial. Thecontents of the vial were stirred magnetically while 1.5 ml of solutionB2 was pipetted into the vial. The vial was stoppered and sealed with analuminum shell. The vial contents were stirred for 16 hours to allowsuspension formation. Aqueous Suspension 2 contained insulinotropin 1mg/ml, protamine base 0.075 mg/ml, and phenol 2.2 mg/ml in PBS. Thissuspension was used for in vivo pharmacokinetic studies in rats.

EXAMPLE 3 Insulinotropin (1 mg/ml) Suspension

Solution A3 Preparation

20 mg of insulinotropin was weighed into a 10 ml volumetric flask.Approximately 8 ml of PBS was added to the flask to disperse anddissolve the drug. The q.s. amount of PBS was added to the flask.Solution A3 was filtered by a syringe through a 0.22μ filter into a 10ml glass vial. Solution A3 contained insulinotropin 2 mg/ml in PBS.

Solution B3 Preparation

8 mg of protamine sulfate, 44 mg of phenol, and 323 mg of glycerin wereweighed into a 10 ml volumetric flask. The q.s. amount of PBS was addedto the flask to dissolve the protamine sulfate, the phenol, and theglycerin. This solution was filtered by a syringe through a 0.22μ filterinto a 10 ml glass vial. Solution B3 contained protamine base 0.6 mg/ml,phenol 4.4 mg/ml, and glycerin 32 mg/ml in PBS.

Aqueous Suspension 3

1.5 ml of Solution A3 was pipetted into a 3.5 ml type I glass vial. Thecontents of the vial were stirred magnetically while 1.5 ml of SolutionB3 was pipetted into the vial. The vial was stoppered and sealed with analuminum shell. The vial contents were stirred for 16 hours to allowsuspension formation. Aqueous Suspension 3 contained insulinotropin 1mg/ml, protamine base 0.3 mg/ml, phenol 2.2 mg/ml, and glycerin 16 mg/mlin PBS. This suspension was used for in vivo pharmacokinetic studies inrats.

EXAMPLE 4 Insulinotropin (1 mg/ml) Suspension

Solution A4 Preparation

20 mg of insulinotropin was weighed into a 10 ml volumetric flask.Approximately 8 ml of PBS was added to the flask to disperse anddissolve the drug. The q.s. amount of PBS was added to the flask.Solution A4 was filtered by a syringe through a 0.22μ filter (MilliporeMillex-GV) into a 10 ml glass vial. Solution A4 contained insulinotropin2 mg/ml in PBS.

Solution B4 Preparation

8 mg of protamine sulfate and 52 mg of m-cresol were weighed into a 10ml volumetric flask. The q.s. amount of PBS was added to the flask todissolve the protamine sulfate and the m-cresol. This solution wasfiltered through a 0.22μ filter into a 10 ml glass vial. Solution B4contained protamine base 0.6 mg/ml and m-cresol 5 mg/ml in PBS.

Aqueous Suspension 4

1.5 ml of solution A4 was pipetted into a 3.5 ml type I glass vial. Thecontents of the vial were stirred magnetically while 1.5 ml of solutionB4 was pipetted into the vial. The vial was stoppered and sealed with analuminum shell. The vial contents were stirred for 16 hours to allowcrystal formation. Aqueous Suspension 4 contained insulinotropin 1mg/ml, protamine base 0.3 mg/ml and m-cresol 2.5 mg/ml in PBS. Thissuspension was used for in vivo pharmacokinetic studies in rats.

EXAMPLE 5 Insulinotropin (1 mg/ml) Suspension

Solution A5 Preparation

50 mg of insulinotropin was weighed into a 25 ml volumetric flask.Approximately 23 ml of PBS was added to the flask to disperse anddissolve the drug. The q.s. amount of PBS was added to the flask.Solution A5 was filtered by a syringe through a 0.22μ filter into a 50ml glass vial. Solution A5 contained insulinotropin 2 mg/ml in PBS.

Phenol Stock Solution Preparation

0.44 g of phenol was weighed into a 100 ml volumetric flask.Approximately 95 ml of PBS was added to the flask to dissolve thephenol. The q.s. amount of PBS was added to the flask to dissolve thephenol. The resulting solution (4.4 mg/ml phenol) was used to prepareSolution B5.

Solution B5 Preparation

Solution B5 was prepared by filtering 25 ml of the phenol stock solutionthrough a 0.2μ filter into a 50 ml glass vial. Solution B5 containedphenol 4.4 mg/ml in PBS.

Aqueous Suspension 5

1.25 ml of solution A5 was pipetted into a 3.5 ml type I glass vial. Thecontents of the vial were stirred magnetically while 1.25 ml of solutionB5 was pipetted into the vial. The vial was stoppered and sealed with analuminum shell. The vial contents were stirred for 16 hours to allowsuspension formation. Aqueous Suspension 5 contained insulinotropin 1mg/ml and phenol 2.2 mg/ml in PBS. This suspension was used for in vivopharmacokinetic studies in rats.

EXAMPLE 6 Insulinotropin (1 mg/ml) Suspension

Solution A6 Preparation

50 mg of insulinotropin was weighed into a 25 ml volumetric flask.Approximately 23 ml of PBS was added to the flask to disperse anddissolve the drug. The q.s. amount of PBS was added to the flask.Solution A6 was filtered by a syringe through a 0.22μ filter into a 50ml glass vial. Solution A6 contained insulinotropin 2 mg/ml in PBS.

Phenol Stock Solution Preparation

0.44 g of phenol was weighed into a 100 ml volumetric flask.Approximately 95 ml of PBS was added to the flask to dissolve thephenol. The q.s. amount of PBS was added to the flask to dissolve thephenol. The resulting solution (4.4 mg/ml phenol) was used to prepareSolution B6.

Solution B6 Preparation

Solution B6 was prepared by weighing 1.25 mg of protamine sulfate into a25 ml volumetric flask. Approximately 20 ml of phenol stock solution wasadded to the flask to dissolve the protamine sulfate. The q.s. amount ofphenol stock solution was added to the flask. Solution B6 was filteredthrough a 0.22μ filter into a 50 ml glass vial. Solution B6 containedphenol 4.4 mg/ml and protamine base 0.038 mg/ml in PBS.

Aqueous Suspension 6

1.25 ml of solution A6 was pipetted into a 3.5 ml type I glass vial. Thecontents of the vial were stirred magnetically while 1.25 ml of solutionB6 was pipetted into the vial. The vial was stoppered and sealed with analuminum shell. The vial contents were stirred for 16 hours to allowsuspension formation. Aqueous Suspension 6 contained insulinotropin 1mg/ml, phenol 2.2 mg/ml, and protamine base 0.019 mg/ml in PBS. Thissuspension was used for in vivo pharmacokinetic studies in rats.

EXAMPLE 7 Insulinotropin (1 mg/ml) Suspension

Solution A7 Preparation

50 mg of insulinotropin was weighed into a 25 ml volumetric flask.Approximately 23 ml of PBS was added to the flask to disperse anddissolve the drug. The q.s. amount of PBS was added to the flask.Solution A7 was filtered by a syringe through a 0.22μ filter into a 50ml glass vial. Solution A7 contained insulinotropin 2 mg/ml in PBS.

Phenol Stock Solution Preparation

0.44 g of phenol was weighed into a 100 ml volumetric flask.Approximately 95 ml of PBS was added to the flask to dissolve thephenol. The q.s. amount of PBS was added to the flask to dissolve thephenol. The resulting solution (4.4 mg/ml phenol) was used to prepareSolution B7.

Solution B7 Preparation

Solution B7 was prepared by weighing 2.5 mg of protamine sulfate into a25 ml volumetric flask. Approximately 20 ml of phenol stock solution wasadded to the flask to dissolve the protamine sulfate. The q.s. amount ofphenol stock solution was added to the flask. Solution B7 was filteredthrough a 0.22μ filter into a 50 ml glass vial. Solution B7 containedphenol 4.4 mg/ml and protamine base 0.075 mg/ml in PBS.

Aqueous Suspension 7

1.25 ml of solution A7 was pipetted into a 3.5 ml type I glass vial. Thecontents of the vial were stirred magnetically while 1.25 ml of solutionB7 was pipetted into the vial. The vial was stoppered and sealed with analuminum shell. The vial contents were stirred for 16 hours to allowsuspension formation. Aqueous Suspension 7 contained insulinotropin 1mg/ml, phenol 2.2 mg/ml, and protamine base 0.038 mg/ml in PBS. Thissuspension was used for in vivo pharmacokinetic studies in rats.

EXAMPLE 8 Insulinotropin (1 mg/ml) Suspension

Solution A12 Preparation

20 mg of insulinotropin was weighed into a 10 ml volumetric flask.Approximately 8 ml of PBS was added to the flask to disperse anddissolve the drug. The q.s. amount of PBS was added to the flask.Solution A12 was filtered by a syringe through a 0.22μ filter into a 10ml glass vial. Solution A12 contained insulinotropin 2 mg/ml in PBS.

Solution B12

Solution B12 was prepared by weighing 20 mg of phenol into a 10 mlvolumetric flask. Approximately 8 ml of PBS was added to the flask todissolve the phenol. The q.s. amount of PBS was added to the flask.Solution B12 was filtered through a 0.22μ filter into a 10 ml glassvial. Solution B12 contained phenol 2 mg/ml in PBS.

Aqueous Suspension 12

4 ml of solution A12 was pipetted into a 10 ml type I glass vial. Thecontents of the vial were stirred while 4 ml of solution B12 waspipetted into the vial. The vial was stoppered and sealed with analuminum shell. The vial contents were stirred for 16 hours to allowsuspension formation. Aqueous Suspension 12 contained insulinotropin 1mg/ml and phenol 1 mg/ml in PBS. This suspension was used for in vivopharmacokinetic studies in rats.

EXAMPLE 9 Insulinotropin (1 mg/ml) Suspension

Solution A15 Preparation

20 mg of insulinotropin was weighed into a 10 ml volumetric flask.Approximately 8 ml of phosphate buffer (PB) was added to the flask todissolve the drug. The q.s. amount of PB was added to the flask.Solution A15 was filtered by a syringe through a 0.22μ filter into a 10ml glass vial. Solution A15 contained insulinotropin 2 mg/ml in PB.

Solution B15 Preparation

Solution B15 was prepared by weighing 8 mg of protamine sulfate into a10 ml volumetric flask. Approximately 8 ml of PB was added to the flaskto dissolve the protamine sulfate. The q.s. amount of PB was added tothe flask. Solution B15 was filtered through a 0.22μ filter into a 10 mlglass vial. Solution B15 contained protamine base 0.6 mg/ml in PBS.

Aqueous Suspension 15

3 ml of solution A15 was pipetted into a 10 ml type I glass vial. Thecontents of the vial were stirred while 3 ml of solution B15 waspipetted into the vial. The vial was stoppered and sealed with analuminum shell. The vial contents were stirred for 16 hours to allowsuspension formation. Aqueous Suspension 15 contained insulinotropin 1mg/ml and protamine base 0.3 mg/ml in PB. This suspension was used forIn vivo pharmacokinetic studies in rats.

EXAMPLE 10 Insulinotropin (1 mg/ml) Suspension

Solution A16 Preparation

20 mg of insulinotropin was weighed into a 10 ml volumetric flask.Approximately 8 ml of PB was added to the flask to dissolve the drug.The q.s. amount of PB was added to the flask. Solution A16 was filteredby a syringe through a 0.22μ filter into a 10 ml glass vial. SolutionA16 contained insulinotropin 2 mg/ml in PB.

Solution B16 Preparation

Solution B16 was prepared by weighing 44 mg of phenol into a 10 mlvolumetric flask. Approximately 8 ml of PB was added to the flask todissolve the phenol. The q.s. amount of PB was added to the flask.Solution B16 was filtered through a 0.22μ filter into a 10 ml glassvial. Solution B16 contained phenol 4.4 mg/ml in PB.

Aqueous Suspension 16

3 ml of Solution A16 was pipetted into a 10 ml type I glass vial. Thecontents of the vial were stirred magnetically while 3 ml of SolutionB16 was pipetted into the vial. The vial was stoppered and sealed withan aluminum shell. The vial contents were stirred for 16 hours to allowsuspension formation. Aqueous Suspension 16 contained insulinotropin 1mg/ml and phenol 2.2 mg in PB. This suspension was used for in vivopharmacokinetic studies in rats.

EXAMPLE 11 Insulinotropin (1 mg/ml) Suspension

Aqueous Suspension 17

10 mg of insulinotropin was weighed into a 10 ml volumetric flask.Approximately 8 ml of PB was added to the flask to dissolve the drug.The q.s. amount of PB was added to the flask. The contents of the flaskwas filtered by syringe through a 0.22μ filter into a 10 ml type I glassvial. The vial was stoppered and sealed with an aluminum shell. The vialcontents were stirred for 16 hours to allow suspension formation.Aqueous Suspension 17 contained insulinotropin 1 mg/ml in PB. Thissuspension was used for in vivo pharmacokinetic studies in rats.

EXAMPLE 12 Insulinotropin (1 mg/ml) Suspension

Aqueous Suspension 18

10 mg of insulinotropin was weighed into a 10 ml volumetric flask.Approximately 8 ml of PBS was added to the flask to dissolve the drug.The q.s. amount of PBS was added to the flask. The contents of the flaskwere filtered by a syringe through a 0.22μ filter into a 10 ml type Iglass vial. The vial was stoppered and sealed with an aluminum shell.The vial contents were stirred gently (making sure no foam or bubbleformed) for 16 hours to allow suspension formation. Aqueous Suspension18 contained insulinotropin 1 mg/ml in PBS. This suspension was used forin vivo pharmacokinetic studies in rats.

EXAMPLE 13 Insulinotropin (0.2 mg/ml) Suspension

Solution A22 Preparation

Solution A22 was prepared by weighing 2 mg of insulinotropin into a 5 mlvolumetric flask. Approximately 3 ml of PBS was added to the flask todissolve lhe drug. The q.s. amount of PBS was added to the flask.Solution A22 was filtered by a syringe through a 0.22μ filter into a 10ml glass vial. Solution A22 contained insulinotropin 0.4 mg/ml in PBS.

Solution B22 Preparation

Solution B22 was prepared by weighing 44 mg of phenol into a 10 mlvolumetric flask. Approximately 8 ml of PBS was added to the flask todissolve the phenol. The q.s. amount of PBS was added to the flask.Solution B22 was filtered through a 0.22μ filter into a 10 ml glassvial. Solution B22 contained phenol 4.4 mg/ml in PBS.

Aqueous Suspension 22

1.5 ml of solution A22 was pipetted into a 3.5 ml type I glass vial. Thecontents of the vial were stirred magnetically while 1.5 ml of solutionB22 was pipetted into the vial. The vial was stoppered and sealed withan aluminum shell. The vial contents were stirred for 16 hours to allowsuspension formation. Aqueous Suspension 22 contained insulinotropin 0.2mg/ml and phenol 2.2 mg/ml in PBS. This suspension was used for in vivopharmacokinetic studies in rats.

EXAMPLE 14 Insulinotropin (0.2 mg/ml) Suspension

Solution A23 Preparation

Solution A23 was prepared by weighing 2 mg of insulinotropin into a 5 mlvolumetric flask. Approximately 3 ml of PBS was added to the flask todissolve the drug. The q.s. amount of PBS was added to the flask.Solution A23 was filtered by a syringe through a 0.22μ filter into a 10ml glass vial. Solution A23 contained insulinotropin 0.4 mg/ml in PBS.

Solution B23 Preparation

Solution B23 was prepared by weighing 8.8 mg of phenol into a 10 mlvolumetric flask. Approximately 8 ml of PBS was added to the flask todissolve the phenol. The q.s. amount of PBS was added to the flask.Solution B23 was filtered through a 0.22μ filter into a 10 ml glassvial. Solution B23 contained phenol 0.88 mg/ml in PBS.

Aqueous Suspension 23

1.5 ml of solution A23 was pipetted into a 3.5 ml type I glass vial. Thecontents of the vial were stirred magnetically while 1.5 ml of solutionB23 was pipetted into tie vial. The vial was stoppered and sealed withan aluminum shell. The vial contents were stirred for 16 hours to allowsuspension formation. Aqueous Suspension 23 contained insulinotropin 0.2mg/ml and phenol 0.44 mg/ml in PBS. This suspension was used for in vivopharmacokinetic studies in rats.

EXAMPLE 15 Insulinotropin (1 mg/ml) Suspension

Solution A24 Preparation

Solution A24 was prepared by weighing 10 mg of insulinotropin into a 5ml volumetric flask. Approximately 3 ml of PBS was added to the flask todissolve the drug. The q.s. amount of PBS was added to the flask.Solution A24 was filtered by a syringe through a 0.22μ filter into a 10ml glass vial. Solution A24 contained insulinotropin 2 mg/ml in PBS.

Solution B24 Preparation

Solution B24 was prepared by weighing 8 mg of protamine sulfate into a10 ml volumetric flask. Approximately 8 ml of PBS was added to the flaskto dissolve the protamine sulfate. The q.s. amount of PBS was added tothe flask. Solution B24 was filtered through a 0.22μ filter into a 10 mlglass vial. Solution B24 contained protamine base 0.6 mg/ml in PBS.

Aqueous Suspension 24

1.5 ml of solution A24 was pipetted into a 3.5 ml type I glass vial. Thecontents of the vial were stirred magnetically while 1.5 ml of solutionB24 was pipetted into the vial. The vial was stoppered and sealed withan aluminum shell. The vial contents were stirred for 16 hours to allowsuspension formation. Aqueous Suspension 24 contained insulinotropin 1mg/ml and protamine base 0.3 mg/ml in PBS. This suspension was used forin vivo pharmacokinetic studies in rats.

EXAMPLE 16 Insulinotropin (1 mg/ml) Suspension

Solution A25 Preparation

Solution A25 was prepared by weighing 10 mg of insulinotropin into a 5ml volumetric flask. Approximately 3 ml of PBS was added to the flask todissolve the drug. The q.s. amount of PBS was added to the flask.Solution A25 was filtered by a syringe through a 0.22μ filter into a 10ml glass vial. Solution A25 contained insulinotropin 2 mg/ml in PBS.

Solution B25 Preparation

Solution B25 was prepared by weighing 53 mg of m-cresol into a 10 mlvolumetric flask. Approximately 8 ml of PBS was added to the flask todissolve the m-cresol. The q.s. amount of PBS was added to the flask.Solution B25 was filtered through a 0.22μ filter into a 10 ml glassvial. Solution B25 contained m-cresol 5.3 mg/ml in PBS.

Aqueous Suspension 25

1.5 ml of solution A25 was pipetted into a 3.5 ml type I glass vial. Thecontents of the vial were stirred magnetically while 1.5 ml of solutionB25 was pipetted into the vial. The vial was stoppered and sealed withan aluminum shell. The vial contents were stirred for 16 hours to allowsuspension formation. Aqueous Suspension 25 contained insulinotropin 1mg/ml and m-cresol 2.5 mg/ml in PBS. This suspension was used for invivo pharmacokinetic studies in rats.

EXAMPLE 17 Insulinotropin (0.5 mg/ml) Suspension

Solution A29 Preparation

Solution A29 was prepared by weighing 25 mg of insulinotropin into a 25ml volumetric flask. Approximately 20 ml of PBS was added to the flaskto dissolve the drug. The q.s. amount of PBS was added to the flask.Solution A29 was filtered by a syringe through a 0.22μ filter into a 50ml glass vial. Solution A29 contained insulinotropin 1 mg/ml in PBS.

Solution B29 Preparation

Solution B29 was prepared by weighing 50 mg of phenol into a 50 mlvolumetric flask. Approximately 40 ml of PBS was added to the flask todissolve the phenol. The q.s. amount of PBS was added to the flask.Solution B29 was filtered through a 0.22μ filter into a 50 ml glassvial. Solution B29 contained phenol 1.0 mg/ml in PBS.

Aqueous Suspension 29

1.5 ml of solution A29 was pipetted into a 3.5 ml type I glass vial. Thecontents of the vial were stirred magnetically while 1.5 ml of solutionB29 was pipetted into the vial. The vial was stoppered and sealed withan aluminum shell. The vial contents were stirred for 16 hours to allowsuspension formation. Aqueous Suspension 29 contained insulinotropin 0.5mg/ml and phenol 0,5 mg/ml in PBS. This suspension was used for in vivopharmacokinetic studies in rats.

EXAMPLE 18 Insulinotropin (1 mg/ml) Suspension

Solution A31 Preparation

10 mg of insulinotropin was weighed into a 5 ml volumetric flask.Approximately 4 ml of PBS was added to the flask to disperse anddissolve the drug. The q.s. amount of PBS was added to the flask.Solution A31 was filtered by a syringe through a 0.22μ filter into a 10ml glass vial. Solution A31 contained insulinotropin 2 mg/ml in PBS.

Solution B31 Preparation

Solution B31 was prepared by weighing 50 mg of phenol into a 50 mlvolumetric flask. Approximately 40 ml of PBS was added to the flask todissolve the phenol. The q.s. amount of PBS was added to the flask.Solution B31 was filtered through a 0.22μ filter into a 50 ml glassvial. Solution B31 contained phenol 1 mg/ml in PBS.

Aqueous Suspension 31

1.5 ml of solution A31 was pipetted into a 3.5 ml type I glass vial. Thecontents of the vial were stirred magnetically while 1.5 ml of solutionB31 was pipetted into the vial. The vial was stoppered and sealed withan aluminum shell. The vial contents were stirred for 16 hours to allowsuspension formation. Aqueous Suspension 31 contained insulinotropin 1mg/ml and phenol 0.5 mg/ml in PBS. This suspension was used for in vivopharmacokinetic studies in rats.

EXAMPLE 19 Insulinotropin (4 mg/mL) Suspension

Solution A51 Preparation

22.2 mg of insulinotropin was weighed into a 10 mL glass vial. 5 mL ofPBS was pipetted into the vial to dissolve the drug. This solution wasfiltered through a 0.22μ filter (low protein binding) into a 10 mL glassvial. Solution A51 contained insulinotropin 4.44 mg/mL in PBS.

Solution B51 Preparation

110 mg of phenol and 30 mg of protamine sulfate were weighed into a 5 mLvolumetric flask. Approximately 4 mL of PBS was added to the flask todissolve the phenol and protamine sulfate. The flask was filled to themark with PBS. The solution was filtered through a 0.22μ filter (lowprotein binding) into a 10 mL glass vial. Solution B51 contained phenol22 mg/mL and protamine base 4.5 mg/mL in PBS.

Aqueous Suspension 51

3 mL of Solution A51 and 0.33 mL of Solution B51 were pipetted into a3.5 mL type I glass vial. The contents of the vial were shaken gently toensure a homogeneous mix. The vial was allowed to sit at ambienttemperature for 16 hours. Aqueous Suspension 51 contained insulinotropin4 mg/mL, protamine base 0.44 mg/mL, and phenol 2.2 mg/mL in PBS. Thissuspension was used for in vivo pharmacokinetic studies in rats

EXAMPLE 20 Insulinotropin (4 mg/mL) Suspension

Solution A52 Preparation

22.2 mg of insulinotropin was weighed into a 10 mL glass vial. 5 mL ofPBS was pipetted into the vial to dissolve the drug. This solution wasfiltered through a 0.22μ filter (low protein binding) into a 10 mL glassvial. Solution A52 contained insulinotropin 4.44 mg/mL in PBS.

Solution B52 Preparation

110 mg of phenol and 15.6 mg of zinc acetate dihydrate were weighed intoa 5 mL volumetric flask. Approximately 4 mL of water for injection wasadded to the flask to dissolve the phenol and zinc acetate dihydrate.The flask was filled to the mark with water for injection. The solutionwas filtered through a 0.22μ filter (low protein binding) into a 10 mLglass vial. Solution B52 contained phenol 22 mg/mL and zinc acetatedihydrate 7.8 mg/mL in water for injection.

Aqueous Suspension 52

3 mL of Solution A52 and 0.33 mL of Solution 652 were pipetted into a3.5 mL type I glass vial. The contents of the vial were shaken gently toensure a homogeneous mix. The vial was allowed to sit at ambienttemperature for 16 hours. Aqueous Suspension 52 contained insulinotropin4 mg/mL, zinc acetate dihydrate 0.78 mg/mL, and phenol 2.2 mg/mL in PBS.This suspension was used for in vivo pharmacokinetic studies in rats.

EXAMPLE 21 Insulinotropin (4 mg/mL) Suspension

Phenol Solution Preparation

244 mg of phenol was weighed into a 100 mL volumetric flask.Approximately 90 mL of water for injection was added to the flask todissolve the phenol. The flask was filled to the mark with water forinjection. The pH of this solution was adjusted to pH 9.0 with 5% NaOHsolution. The Phenol Solution contained phenol 2.44 mg/mL in water forinjection pH 9.0.

Solution A71 Preparation

22.2 mg of insulinotropin was weighed into a 10 mL glass vial. 5 mL ofthe Phenol Solution was pipetted into the vial to dissolve the drug.This solution was filtered through a 0.22μ filter (low protein binding)into a 10 mL glass vial. Solution A71 contained insulinotropin 4.44mg/mL and phenol 2.44 mg/mL in water for injection.

Solution B71 Preparation

116 mg of protamine sulfate was weighed into a 10 mL volumetric flask.Approximately 8 mL of water for injection was added to the flask todissolve the protamine sulfate. The flask was filled to the mark withwater for injection. The solution was filtered through a 0.22μ filter(low protein binding) into a 10 mL glass vial. Solution B71 containedprotamine base 8.7 mg/mL in water for injection.

Solution C71 Preparation

156 mg of zinc acetate dihydrate and 1.632 g of NaCl were weighed into a10 mL volumetric flask. Approximately 8 mL of water for injection wasadded to the flask to dissolve the zinc acetate dihydrate and NaCl. Theflask was filled to the mark with water for injection. The solution wasfiltered through a 0.22μ filter (low protein binding) into a 10 mL glassvial. Solution C71 contained zinc acetate dihydrate 15.6 mg/mL and NaCl163.2 mg/mL in water for injection.

Aqueous Suspension 71

3 mL of Solution A71, 0.165 mL of Solution B71, and 0.165 mL of SolutionC71 were pipetted into a 3.5 mL type I glass vial. The contents of thevial were shaken gently to ensure a homogeneous mix. The vial wasallowed to sit at ambient temperature for 16 hours. Aqueous Suspension71 contained insulinotropin 4 mg/mL, protamine base 0.435 mg/mL, zincacetate dihydrate 0.78 mg/mL, NaCl 8.16 mg/mL, and phenol 2.2 mg/mL inwater for injection. This suspension was used for in vivopharmacokinetic studies in rats.

EXAMPLE 22 Insulinotropin (4 mg/mL) Suspension

m-Cresol Solution Preparation

244 mg of m-cresol was weighed into a 100 mL volumetric flask.Approximately 90 mL of water for injection was added to the flask todissolve the m-cresol. The flask was filled to the mark with water forinjection. The pH of this solution was adjusted to pH 9.0 with 5% NaOHsolution. The m-cresol Solution contained m-cresol 2.44 mg/mL in waterfor injection pH 9.0.

Solution A100 Preparation

22.2 mg of insulinotropin was weighed into a 10 mL glass vial. 5 mL ofthe m-cresol Solution was pipetted into the vial to dissolve the drug.This solution was filtered through a 0.22 μ filter (low protein binding)into a 10 mL glass vial. Solution A100 contained insulinotropin 4.44mg/mL and m-cresol 2.44 mg/mL in water for injection.

Solution B100 Preparation

116 mg of protamine sulfate was weighed into a 10 mL volumetric flask.Approximately 8 mL of water for injection was added to the flask todissolve the protamine sulfate. The flask was filled to the mark withwater for injection. The solution was filtered through a 0.22μ filter(low protein binding) into a 10 mL glass vial. Solution B100 containedprotamine base 8.7 mg/mL in water for injection.

Solution C100 Preparation

156 mg of zinc acetate dihydrate and 1.632 g of NaCl were weighed into a10 mL volumetric flask. Approximately 8 mL of water for injection wasadded to the flask to dissolve the zinc acetate dihydrate and NaCl. Theflask was filled to the mark with water for injection. The solution wasfiltered through a 0.22μ filter (low protein binding) into a 10 mL glassvial. Solution C100 contained zinc acetate dihydrate 15.6 mg/mL and NaCl163.2 mg/mL in water for injection.

Aqueous Suspension 100

3 mL of Solution A100, 0.165 mL of Solution B100, and 0.165 mL ofSolution C100 were pipetted into a 3.5 mL type I glass vial. Thecontents of the vial were shaken gently to ensure a homogeneous mix. Thevial was allowed to sit at ambient temperature for 16 hours. AqueousSuspension 100 contained insulinotropin 4 mg/mL, protamine base 0.435mg/mL, zinc acetate dihydrate 0.78 mg/mL, NaCl 8.16 mg/mL, and m-cresol2.2 mg/mL in water for injection. This suspension was used for in vivopharmacokinetic studies in rats.

EXAMPLE 23 Insulinotropin (4 mg/mL) Suspension

Solution A68 Preparation

22.2 mg of insulinotropin was weighed into a 10 mL glass vial. 5 mL ofthe PBS was pipetted into the vial to dissolve the drug. This solutionwas filtered through a 0.22μ filter (low protein binding) into a 10 mLglass vial. Solution A68 contained insulinotropin 4.44 mg/mL in PBS.

Solution B68 Preparation

116 mg of protamine sulfate was weighed into a 10 mL volumetric flask.Approximately 8 mL of water for injection was added to the flask todissolve the protamine sulfate. The flask was filled to the mark withwater for injection. The solution was filtered through a 0.22μ filter(low protein binding) into a 10 mL glass vial. Solution B68 containedprotamine base 8.7 mg/mL in water for injection.

Solution C68 Preparation

156 mg of zinc acetate dihydrate and 440 mg of phenol was weighed into a10 mL volumetric flask. Approximately 8 mL of water for injection wasadded to the flask to dissolve the zinc acetate dihydrate and phenol.The flask was filled to the mark with water for injection. The solutionwas filtered through a 0.22μ filter (low protein binding) into a 10 mLglass vial. Solution C68 contained zinc acetate dihydrate 15.6 mg/mL andphenol 44 mg/mL in water for injection.

Aqueous Suspension 68

3 mL of Solution A68, 0.165 mL of Solution B68, and 0.165 mL of SolutionC68 were pipetted into a 3.5 mL type I glass vial. The contents of thevial were shaken gently to ensure a homogeneous mix. The vial wasallowed to sit at ambient temperature for 16 hours. Aqueous Suspension68 contained insulinotropin 4 mg/mL, protamine base 0.435 mg/mL, zincacetate dihydrate 0.78 mg/mL, and phenol 2.2 mg/mL in PBS. Thissuspension was used for in vivo pharmacokinetic studies in rats.

EXAMPLE 24 Insulinotropin (4 mg/mL) Suspension

Solution A67 Preparation

22.2 mg of insulinotropin was weighed into a 10 mL glass vial. 5 mL ofthe PBS was pipetted into the vial to dissolve the drug. This solutionwas filtered through a 0.22μ filter (low protein binding) into a 10 mLglass vial. Solution A67 contained insulinotropin 4.44 mg/mL in PBS.

Solution B67 Preparation

116 mg of protamine sulfate was weighed into a 10 mL volumetric flask.Approximately 8 mL of water for injection was added to the flask todissolve the protamine sulfate. The flask was filled to the mark withwater for injection. The solution was filtered through a 0.22μ filter(low protein binding) into a 10 mL glass vial. Solution B67 containedprotamine base 8.7 mg/mL in water for injection.

Solution C67 Preparation

156 mg of zinc acetate dihydrate and 440 mg of m-cresol were weighedinto a 10 mL volumetric flask. Approximately 8 mL of water for injectionwas added to the flask to dissolve the zinc acetate dihydrate andm-cresol. The flask was filled to the mark with water for injection. Thesolution was filtered through a 0.22μ filter (low protein binding) intoa 10 mL glass vial. Solution C67 contained zinc acetate dihydrate 15.6mg/mL and m-cresol 44 mg/mL in water for injection.

Aqueous Suspension 67

3 mL of Solution A67, 0.165 mL of Solution B67, and 0.165 mL of SolutionC67 were pipetted into a 3.5 mL type I glass vial. The contents of thevial were shaken gently to ensure a homogeneous mix. The vial wasallowed to sit at ambient temperature for 16 hours. Aqueous Suspension67 contained insulinotropin 4 mg/mL, protamine base 0.435 mg/mL, zincacetate dihydrate 0.78 mg/mL, and m-cresol 2.2 mg/mL in PBS. Thissuspension was used for in vivo pharmacokinetic studies in rats.

EXAMPLE 25

Solution A39 Preparation

67.6 mg of insulinotropin was weighed into a glass vial. Approximately22 mL of water for injection was added to the vial to dissolve theinsulinotropin. The pH of the vial content was adjusted to 9.6 usingNaOH to make a clear solution. Water for injection was added to the vialto make the final drug concentration to be 2.5 mg/ml.

Solution B39 Preparation

386.8 mg of zinc acetate dihydrate was weighed into a 100 ml volumetricflask. Approximately 80 mL of water for injection was added to the flaskto dissolve the zinc acetate dihydrate. The flask was filled to the markwith water for injection. Solution B39 contained zinc acetate dihydrate3.9 mg/mL in water for injection.

Solution C39 Preparation

1.095 g of phenol was weighed into a 50 ml volumetric flask.Approximately 40 mL of water for injection was added to the flask todissolve the phenol. The flask was filled to the mark with water forinjection. Solution C39 contained phenol 21.9 mg/mL in water forinjection.

Solution D39 Preparation

2.25 g of NaCl was weighed into a 25 mL volumetric flask. Approximately20 mL of Solution C39 was added to the flask to dissolve the NaCl. Theflask was filled to the mark with Solution C39. Solution D39 containedNaCl 9% (w/v) and phenol 21.9 mg/mL in water for injection.

Aqueous Suspension 39

All solutions were filtered through 0.22 μl filters (low proteinbinding). 9 ml of Solution A39 was transferred to a 10 ml sample vial. 1ml of Solution B39 was added to the vial while stirring gently.Precipitates were formed immediately. The pH was measured to be 7.0. Thevial was allowed to sit at ambient temperature for about 18 hours. 4 mlof the sample was transferred to a separate 10 ml vial, and 0.44 ml ofSolution D39 was added to the vial. The sample was stirred gently for 5minutes and was then allowed to sit at ambient temperature overnight.

Aqueous Suspension 39 contained insulinotropin 2 mg/ml, phenol 2.2mg/ml, NaCl 0.9%, and zinc acetate 0.39 mg/ml. This suspension was usedfor in vivo pharmacokinetic studies in rats.

EXAMPLE 26

Solution A53 Preparation

32.5 mg of insulinotropin was weighed into a 10 ml glass vial. 6 ml ofwater for injection was added to the vial. The pH of the vial contentwas adjusted to 9.6 using 1% (w/v) NaOH to make a clear solution.Appropriate amount of water for injection was added to make the drugconcentration to be 5.0 mg/ml.

Solution B53 Preparation

390 mg of zinc acetate dihydrate was weighed into a 50 ml volumetricflask. Approximately 40 mL of water for injection was added to the flaskto dissolve the zinc acetate dihydrate. The flask was filled to the markwith water for injection. Solution B53 contained zinc acetate dihydrate7.8 mg/mL in water for injection.

Aqueous Suspension 53

All solutions were filtered through 0.22μ filters (low protein binding).2.4 mL of Solution A53 was transferred to a 3.5 ml vial. 300 μl ofSolution B53 was added to the vial while stirring gently. Birefringentprecipitates were formed immediately after the addition. The pH wasmeasured to be 6.8. After the vial was allowed to sit at ambienttemperature for 20 hours, 7.5 μl of m-cresol was added directly to thesupernatant of the settled suspension. The suspension was then stirredgently to dissolve the m-cresol. 300 μl of 9% NaCl solution was added tothe suspension with stirring. Aqueous Suspension 53 containedinsulinotropin 4 mg/mL, 0.9% NaCl, 0.78 mg/mL zinc acetate, and 2.5mg/mL m-cresol in water for injection. This suspension was used for invivo pharmacokinetic studies in rats.

EXAMPLE 27

Solution A54 Preparation

32.5 mg of insulinotropin was weighed into a 10 ml glass vial. 6 ml ofwater for injection was added to the vial. The pH of the vial contentwas adjusted to 9.6 using 1% (w/v) NaOH to make a clear solution.Appropriate amount of water for injection was added to make the drugconcentration to be 5.0 mg/ml.

Solution B54 Preparation

390 mg of zinc acetate dihydrate was weighed into a 50 ml volumetricflask. Approximately 40 mL of water for injection was added to the flaskto dissolve the zinc acetate dihydrate. The flask was filled to the markwith water for injection. Solution B54 contained zinc acetate dihydrate7.8 mg/mL in water for injection.

Solution C54 Preparation

1.1 g of phenol and 4.5 g of NaCl were weighed into a 50 ml volumetricflask. Approximately 40 mL of water for injection. The flask was filledto the mark with water for injection. Solution C54 contained phenol 22mg/mL and NaCl 90 mg/mL.

Aqueous Suspension 54

All solutions were filtered through 0.22μ filters (low protein binding).2.4 ml of Solution A54 was transferred to a 3.5 ml vial. 300 μl ofSolution B54 was added to the vial with stirring. Birefringentprecipitates were formed immediately after the addition. The pH wasmeasured to be 6.8. The sample was allowed to sit for 20 hours atambient temperature. 300 μl of Solution C54 was added with gentlestirring. Aqueous Suspension 54 contained insulinotropin 4 mg/mL, zincacetate dihydrate 0.78 mg/mL, phenol 2.2 mg/mL, and NaCl 9 mg/mL inwater for injection. This suspension was used for in vivopharmacokinetic studies in rats.

EXAMPLE 28

Solution A57 Preparation

15 mg of insulinotropin was weighed into a 10 mL glass vial. 3 mL ofwater for injection was added to the vial. The pH of the vial contentwas adjusted to 9.9 using 5% NaOH to dissolve the drug completely.Solution A57 contained insulinotropin 5.0 mg/mL in water for injection.

Solution B57 Preparation

780 mg of zinc acetate dihydrate was weighed into a 100 mL volumetricflask. Approximately 80 mL of water for injection was added to the flaskto dissolve the zinc acetate dihydrate. The flask was filled to the markwith water for injection. Solution B57 contained zinc acetate dihydrate7.8 mg/mL in water for injection.

Solution C57 Preparation

2.2 g of phenol and 9 g of NaCl were weighed into a 100 mL volumetricflask. Approximately 80 mL of water for injection was added to the flaskto dissolve the phenol and the NaCl. The flask was filled to the markwith water for injection. Solution C57 contained phenol 22 mg/ml andNaCl 90 mg/mL in water for injection.

Aqueous Suspension 57

2.4 mL of Solution A57 was transferred to a 3.5 mL vial. The solutionwas stirred gently during addition of 300 μL of Solution B57.Precipitates were formed immediately after the addition of the SolutionB57. The pH was measured and found to be 7.1. The sample was allowed tosit under ambient conditions for 24 hours. 300 μL of Solution C57 wasadded with gentle stirring. Aqueous Suspension 57 containedinsulinotropin 4 mg/mL, zinc acetate dihydrate 0.78 mg/mL, phenol 2.2mg/mL, and NaCl 9 mg/mL in water for injection. This suspension was usedfor in vivo pharmacokinetic studies in rats.

EXAMPLE 29

Solution A64 Preparation

53.3 mg of insulinotropin was weighed into a 30 mL glass vial. Afteradding 11 mL of water for injection, the pH of the vial contents wasadjusted to 8.3 using 5% NaOH (w/v) to dissolve the insulinotropin. ThepH was adjusted down to 6.0 using dilute HCl making sure that thesolution still remained clear. Appropriate amount of water for injectionwas added to make the drug concentration to be 4.4 mg/ml. Solution A64was filtered through a 0.22μ filter (low protein binding) into a 3.5 mLsample vial. 1.8 mL of the filtered solution was transferred to aseparate sterile 3.5 mL vial, and the vial was allowed to sit at ambienttemperature to crystallize for 3 days.

Solution B64 Preparation

780 mg of zinc acetate dihydrate was weighed into a 50 mL volumetricflask. Approximately 40 mL of water for injection was added to the flaskto dissolve the zinc acetate dihydrate. The flask was filled to the markwith water for injection. Solution B64 contained zinc acetate dihydrate15.6 mg/mL in water for injection.

Solution C64 Preparation

18 g of NaCl was weighed into a 100 mL volumetric flask. Approximately80 mL of water for injection was added to the flask to dissolve theNaCl. The flask was filled to the mark with water for injection.Solution C64 contained NaCl 180 mg/mL in water for injection.

Aqueous Suspension 64

After crystallization was completed in Solution A64, 100 μL of SolutionB64 was added to 1.8 mL of the crystal suspension was slow stirring. Thesample was then allowed to sit at ambient temperature for 3 days. 100 μLof Solution C64 was added to the crystal suspension with gentlestirring. The pH of the suspension was adjusted to pH 7.3 using diluteNaOH. 5.0μ of m-cresol was added directly to the pH adjusted crystalsuspension. Aqueous Suspension 64 contained insulinotropin 4 mg/mL, zincacetate dihydrate 0.78 mg/mL, NaCl 9 mg/mL, and m-cresol 2.5 mg/mL inwater for injection. This suspension was used for in vivopharmacokinetic studies in rats.

EXAMPLE 30

Solution A69 Preparation

1 g of NaCl was weighed into a 100 mL volumetric flask. Approximately 80mL of water for injection was added to the flask to dissolve the NaCl.The flask was filled to the mark with water for injection. Solution A69contained NaCl 1% (w/v) in water for injection.

Solution B69 Preparation

390 mg of zinc acetate dihydrate was weighed into a 100 mL volumetricflask. Approximately 80 mL of water for injection was added to the flaskto dissolve the zinc acetate dihydrate. The flask was filled to the markwith water for injection. Solution 669 contained zinc acetate dihydrate3.9 mg/mL in water for injection.

Emulsion C69 Preparation

2.5 mL of sterile filtered (0.22μ low protein binding) m-cresol wastransferred to a 100 mL volumetric flask. The flask was filled withwater for injection to the mark and sonicated to produce a homogenoussuspension. Emulsion C69 contained m-cresol 25 mg/mL in water forinjection.

Aqueous Suspension 69

35.74 mg of insulinotropin was weighed into a 10 mL glass vial. 7 mL ofSolution A69 was added. The pH of the vial contents was adjusted to 9.2to dissolve the drug. The pH of the solution was re-adjusted to 6.5using dilute HCl. Appropriate amount of water for injection was added tomake the drug concentration to be 4.4 mg/ml. The solution was filteredthrough a 0.22μ filter (low protein binding). The solution was allowedto sit at ambient temperature for 6 days, during which insulinotropinwas crystallized. 1.5 mL of the crystal suspension was transferred to aseparate vial. 167 μL of Solution B69 was added with gentle stirring.The sample was allowed to sit at ambient temperature for 1 day. 167 μLof emulsion C69 was added to the supernatant of the settled suspension.The sample was stirred to dissolve the m-cresol. Aqueous Suspension 69contained insulinotropin 3.6 mg/ml, zinc acetate 0.36 mg/ml, NaCl 8.17mg/ml and m-cresol 2.28 mg/ml in water for injection. This suspensionwas used for in vivo pharmacokinetic studies in rats.

EXAMPLE 31

Solution A101 Preparation

10 g of sodium acetate was weighed into a 100 ml volumetric flask.Approximately 80 mL of water for injection was added to the flask todissolve the sodium acetate. The flask was filled to the mark with waterfor injection. Solution A200 contained 100 mg/ml sodium acetate in waterfor injection.

Aqueous Suspension 101

44.4 mg of insulinotropin was weighed into a 10 ml glass vial. 8 ml ofwater for injection was added to the flask. The pH of the vial contentswas adjusted to 9.3 to obtain a clear solution. 1 mL of Solution A200was added to the insulinotropin solution. The pH was then adjusted downto 6.5. The solution was filtered through a 0.22μ filter (low proteinbinding). The filtered solution was allowed to sit at ambienttemperature for 3 days so that crystallization could occur. AqueousSuspension 101 contained insulinotropin 4.9 mg/mL sodium acetate 11.1mg/mL in water for injection. This suspension was used for in vivopharmacokinetic study in rats.

EXAMPLE 32

Solution A82 Preparation

9 g of NaCl was weighed into a 100 mL volumetric flask. Approximately 80mL of water for injection was added to the vial to dissolve the NaCl.The flask was filled to the mark with water for injection. Solution A82contained NaCl 9% (w/v) in water for injection.

Solution B82 Preparation

789 mg of zinc acetate dihydrate was weighed into a 100 mL volumetricflask. Approximately 80 mL of water for injection was added to the vialto dissolve the zinc acetate dihydrate. The flask was filled to the markwith water for injection. Solution B82 contained zinc acetate dihydrate7.89 mg/mL in water for injection.

Emulsion C82 Preparation

2.5 mL of sterile filtered (0.22μ low protein binding) m-cresol wastransferred to a 100 mL volumetric flask. The flask was filled withwater for injection to the mark and sonicated to produce a homogenoussuspension. Emulsion C82 contained m-cresol 25 mg/mL in water forinjection.

Aqueous Suspension 82

All solutions were filtered through 0.22μ filters (low protein binding).45.34 mg of insulinotropin was added to a 10 ml vial to which 8 ml ofwater was added. The pH was adjusted to 9.3 using 5% NaOH. After 1 ml ofSolution A82 was added to the vial, the pH of the solution was adjusteddown to 6.55 using dilute HCl. The solution (5 mg/mL insulinotropin) wasfiltered through a 0.22μ filter (low protein binding). 81 μl of AqueousSuspension 101 (see example 31) was added to the sterile filteredinsulinotropin solution and dispersed by shaking the sample. The samplewas then allowed to sit for 72 hours at ambient temperature to form acrystal suspension. 2.4 ml of the suspension was transferred to a 3.5 mlvial. 300 μl of Solution B82 was added to the vial with gentle stirring.The pH of the vial content was adjusted to 7.3 using dilute NaOH. 300 μlof Emulsion C82 was added to the supernatant of the settled suspension.Aqueous Suspension 82 contained insulinotropin 4 mg/ml, zinc acetatedihydrate 0.79 mg/mL, m-cresol 2.5 mg/mL and 0.9% NaCl in water forinjection. This suspension was used for in vivo pharmacokinetic studiesin rats.

EXAMPLE 33

GLP-1 (7-36) Amide (1 mg/ml) Suspension

Solution A26 Preparation

Solution A26 was prepared by weighing 10 mg of GLP-1 (7-36) Amide into a5 ml volumetric flask. Approximately 3 ml of PBS was added to the flaskto dissolve the drug. The q.s. amount of PBS was added to the flask.Solution A26 was filtered through a 0.22μ filter into a 10 ml glassvial. Solution A26 contained GLP-1 (7-36) 2 mg/ml in PBS.

Solution B26 Preparation

Solution B26 was prepared by weighing 44 mg of phenol into a 10 mlvolumetric flask. Approximately 8 ml of PBS was added to the flask todissolve the phenol. The q.s. amount of PBS was added to the flask.Solution B26 was filtered through a 0.22μ filter into a 10 ml glassvial. Solution B26 contained phenol 4.4 mg/ml in PBS.

Aqueous Suspension 26

1.5 ml of solution A26 was pipetted into a 3.5 ml type I glass vial. Thecontents of the vial were stirred magnetically while 1.5 ml of solutionB26 was pipetted into the vial. The vial was stoppered and sealed withan aluminum shell. The vial contents were stirred gently (making sure nofoam or bubble formed) for 18 hours to allow suspension formation.Aqueous Suspension 26 contained GLP-1 (7-36) Amide 1 mg/ml and phenol2.2 mg/ml in PBS. This suspension was used for in vivo pharmacokineticstudies in rats.

EXAMPLE 34

In one form of the invention, a low solubility form of GLP-1 (7-37) isprepared by combining GLP-1 (7-37) at from 2-15 mg/ml in buffer at pH7-8.5 with a solution of a metal ion salt to obtain solutions with from1-8 mg/ml GLP-1 (7-37) at molar ratios of about 1:1 to 270:1 zinc toGLP-1 (7-37). A heavy precipitate forms and is let stand overnight atroom temperature. The solubility of GLP-1 (7-37) in the metal ionsolution varies with the metal employed. Subsequent measurement of thesolubility of the GLP-1 (7-37) pellet in a non metal-containing solventsuch as PBS or water shows that zinc, cobalt and nickel ions produce lowsolubility forms of GLP-1 (7-37). TABLE 1 Ability of Various metal ionsalts to produce low solubility GLP-1(7-37) Metal ion salt Solubility inmetal sol'n Solubility in PBS Zn Acetate 0.04 μg/ml 0.04 μg/ml ZnChloride 0.04 μg/ml 0.03 μg/ml Co Chloride 0.11 μg/ml 0.04 μg/ml NiSulfate 0.14 μg/ml 0.07 μg/ml Mn Chloride 0.23 μg/ml 1.64 μg/ml MgChloride 1.75 μg/ml no ppt. Ca Chloride 1.98 μg/ml no ppt.Note:In each case, 100 μl of metal ion solution at 5 mM was added to 100 μlGLP-1(7-37) at 5 mg/ml, mixed and allowed to stand overnight. Theinsoluble pellet was removed by centrifugation. The concentration ofGLP-1(7-37) remaining in the metal ion solution was measured. The pelletwas resuspended in phosphate buffered saline (PBS), sonicated andallowed to stand overnight. Again insoluble material was pelleted andGLP-1(7-37) concentration measured.

EXAMPLE 35

Microcrystalline forms of GLP-1 (7-37) can be obtained by mixingsolutions of GLP-1 (7-37) in buffer pH 7-8.5 with certain combinationsof salts and low molecular weight polyethylene glycols (PEG). Table 2describes six specific sets of conditions to produce microcrystallineforms of GLP-1 (7-37). TABLE 2 Selected Reagents Yielding MicrocrystalsReagent # Salt Buffer Precipitant 1 none none 0.4 M K, Na tartrate 2 0.2M Na citrate 0.1 M Tris pH 8.5 30% PEG 400 3 0.2 M MgCl₂ 0.1 M HEPES pH7.5 28% PEG 400 4 0.2 M MgCl₂ 0.1 M HEPES pH 7.5 30% PEG 400 5 0.5 MK₂HPO₄ none 20% PEG 8000 6 none none 30% PEG 1500Note:GLP-1(7-37) stock at 5 mg/ml in 50 mM Tris pH 8.1 was added 1:1 withreagent. Drops were viewed and scored for absence or presence ofinsoluble GLP-1(7-37) in crystalline or amorphous form. In general lowmw PEG's appear to favor crystalline forms. Tris istris(hydroxymethyl)aminomethane and HEPES isN-2-Hydroxyethyl)piperazine-N-2-ethanesulfonic acid.

EXAMPLE 36

Specific combinations of GLP-1 (7-37) and PEG concentrations arerequired to obtain microcrystalline forms and high yields. Table 3 showsspecific combinations of PEG 600 and GLP-1 (7-37) concentrations whichproduce microcrystalline as opposed to amorphous forms of the drug. Theyield of GLP-1 (7-37) in the insoluble form is shown also. TABLE 3Formation/yield of crystalline GLP-1(7-37) 15 22.5% 30% GLP-1(7-37) PEG600 PEG 600 PEG 600 2.0 mg/ml amorphous/8% amorphous/10% amorphous/8%(Form/yield) 3.5 mg/ml crystalline/62% crystalline/26% crystalline/59%(Form/yield) 5.0 mg/ml amorphous/34% crystalline/63% crystalline/72%(Form/yield) 6.5 mg/ml amorphous/52% crystalline/76% crystalline/82%(Form/yield) 8.0 mg/ml amorphous/55 crystalline/82% amorphous/66%(Form/yield) 9.5 mg/ml amorphous/69% crystalline/85% amorphous/83%(Form/yield)Note:Microcrystals of GLP-1(7-37) are prepared by combining solutions ofGLP-1(7-37) at 20 mg/ml in tris buffer at pH 8, 60% polyethylene glycol600 (PEG 600) in H₂O and tris buffer pH 8 to obtain a finalconcentrations of from 15-30% PEG and from 3-10 mg/ml GLP-1. Afterstanding overnight, microcrystals of GLP-1(7-37) form in the solutionwith yields from 50-85%.

EXAMPLE 37

This experiment exemplifies another form of the invention which involvestreating preformed microcrystals of GLP-1 (7-37) with various metal ionsto produce low solubility microcrystalline forms. Microcrystals of GLP-1(7-37) prepared at 8 mg/ml GLP-1 (7-37) and 22.5% PEG as described inExample 22 have a solubility equivalent to pure lyophilized GLP-1(7-37). In order to impart the desired property of low solubility forlong-acting drug delivery, these preformed microcrystals can be treatedwith solutions of metal salts at ratios of metal:GLP-1 (7-37) of from1:1 to 260:1 overnight at room temp. The excess metal salt was removedby a centrifugation/washing process. Table 4 shows the results withseveral divalent cation metal salts as treatment. TABLE 4 Solubility ofGLP-1(7-37) Crystals with Various Treatments GLP-1(7-37) GLP-1(7-37)(mg/ml) in GLP-1(7-37) (mg/ml) in Additive treatment sol'n (mg/ml) inPBS PBS/EDTA None (PBS) 1.2 1.2 ND Citrate pH 5.2 0.15 ND ND ZnCl₂ pH5.2 0.03 0.03 1.1 ZnAc pH 5.2 0.01 0.02 1.1 ZnAc pH 6.5 0.06 0.02 0.92MgSO₄ pH 5.2 0.50 0.55 ND NiSO₄ pH 5.2 0.10 0.04 0.45 MnCl₂ pH 5.2 0.100.10 ND CaCl₂ pH 5.2 0.40 0.27 NDNote:GLP-1(7-37) crystals are grown from a solution of 8 mg/ml IST in 50 mMTris pH 8 with 22.5% PEG 600 added in H₂O. All additive treatmentsolutions are 100 mM divalent ion salt in 10 mM Na citrate pH 5.2 or NaMES pH 6.5.

EXAMPLE 38

Using the methods described herein, both amorphous and microcrystallinelow solubility formulations were prepared using zinc acetate.Subcutaneous injections were made in rats (three animals performulation) and plasma levels of GLP-1 (7-37) were measured byradioimmune assay over 24 hours. FIG. 8 shows the extended duration ofthe drug in plasma compared to a subcutaneous control injection ofsoluble GLP-1 (7-37).

EXAMPLE 39

-   -   45% w/v Polyethylene Glycol 3350 (PEG)    -   1 mg/ml Insulinotropin    -   20 mM Phosphate Buffer    -   qs Sterile Water for Injection (SWFI)

A 50% w/w PEG solution was prepared using SWFI. A 200 mM phosphatebuffer was separately prepared with anhydrous sodium phosphate dibasic(26.85 mg/ml) and sodium phosphate monobasic monohydrate (1.41 mg/ml).If necessary, the pH of the buffer solution was brought to pH 8 witheither sodium hydroxide or hydrochloric acid. The appropriate amount ofinsulinotropin was dissolved in enough of the buffer solution to make a10 mg/ml solution of insulinotropin. The appropriate weight of the PEGsolution was added to the insulinotropin solution, and a sufficientquantity of SWFI was used to bring the solution to the desired volume.The final solution was then sterile filtered with 0.2% filter andaseptically filled into vials. The solution (0.5 ml) was injectedsubcutaneously in rats, and plasma insulinotropin levels followed by RIAassay.

EXAMPLE 40

1.32% w/v Hydroxyethyl Cellulose (HEC)

-   -   1 mg/ml Insulinotropin    -   20 mM Phosphate Buffer    -   100 mM Sodium Chloride    -   qs Sterile Water For Injection (SWFI)

A 2% w/w hydroxethyl cellulose solution was prepared using SWFI. A 200mM phosphate buffer was separately prepared with anhydrous sodiumphosphate dibasic (26.85 mg/ml) and sodium phosphate monobasicmonohydrate (1.41 mg/ml). If necessary, the pH of the buffer solutionwas brought to pH 8 with either sodium hydroxide or hydrochloric acid.The appropriate amount of insulinotropin and sodium chloride weredissolved in enough of the buffer solution to make a 10 mg/ml solutionof insulinotropin. The appropriate weight of the HEC solution was addedto the insulinotropin solution, and a sufficient quantity of SWFI wasused to bring the solution to the desired volume. The final solution wasthen sterile filtered with a 0.2μ filter and aseptically filled intovials. The solution (0.5 ml) was injected subcutaneously in rats, andplasma insulinotropin followed by RIA assay.

EXAMPLE 41

-   -   18% w/v Pluronic F127    -   1 mg/ml Insulinotropin    -   20 mM Phosphate Buffer    -   qs Sterile Water For Injection (SWFI)

A 20% W/W Pluronic F 127 solution was prepared using SWFI. A Polytron(probe homogenizer) with an ice bath was used to dissolve the polymer. A200 mM phosphate buffer was separately prepared with anhydrous sodiumphosphate dibasic (26.85 mg/ml) and sodium phosphate monobasicmonohydrate (1.41 mg/ml). If necessary, the pH of the buffer solutionwas brought to pH 8 with either sodium hydroxide or hydrochloric acid.The appropriate amount of insulinotropin was dissolve in enough of thebuffer solution to make a 10 mg/ml solution of insulinotropin. Theappropriate weight of the Pluronic solution was added to theinsulinotropin solution, and a sufficient quantity of SWFI was used tobring the solution to the desired volume. The final solution was thensterile filtered with a 0.2μ filter and aseptically filled into vials.The solution (0.5 ml) was injected subcutaneously in rats, and plasmainsulinotropin levels followed by RIA assay.

EXAMPLE 42

-   -   Peanut Oil Suspension (Ball Milled)    -   1 mg/ml Insulinotropin    -   1% Tween 80

Tween 80 was added at 1% level to peanut oil. This solution was sterilefiltered with a 0.2 μm filter. Solid insulinotropin was then suspendedin the oil. The particle size was reduced by ball milling with aSzesvari Attritor at 40 RPM for 18 hours (cold water jacket). Thissuspension was then filled into vials. The suspension (0.5 ml) wasinjected subcutaneously in rats, and plasma insulinotropin levelsfollowed by RIA assay.

EXAMPLE 43

-   -   22.6% w/v Dextran    -   1 mg/ml Insulinotropin    -   20 mM Phosphate Buffer    -   qs Sterile Water for Injection

A 50% w/w Dextran solution was prepared using SWFI. A 200 mM phosphatebuffer was separately prepared with anhydrous sodium phosphate dibasic(26.85 mg/ml) and sodium phosphate monobasic monohydrate (1.41 mg/ml).If necessary, the pH of the buffer solution was brought to pH 8 witheither sodium hydroxide or hydrochloric acid. The appropriate amount ofinsulinotropin was dissolved in enough of the buffer solution to make5.0 mg/ml solution of insulinotropin. The appropriate weight of thedextran solution was added to the insulinotropin solution, and asufficient quantity of SWFI was used to bring the solution to thedesired volume. The final solution was then sterile filtered with 0.2 μmfilter and aseptically filled into vials. The solution (0.5 ml) wasinjected subcutaneously into rats, and plasma insulinotropin levels werefollowed by RIA assay.

EXAMPLE 44

Insulinotropin was crystallized from the mixture of phosphate bufferedsaline (PBS), ethanol, and m-cresol. A homogeneous insulinotropin slurry(10 mg/ml) was made with PBS in a glass vial, and a large volume ofethanol (9 times as much as the slurry) was added to the vial while thevial content was stirred magnetically. Very fine amorphous particles ofinsulinotropin formed. m-Cresol was added to the vial so that theresulting m-cresol concentration was 1% (v/v). The vial was capped toprevent solvent from evaporating. The crystallization mixture was storedat room temperature for a couple of days. Needle shape crystallineplates grew from the amorphous particles. The lengths of the crystalsare between 50 and 200 μm, and widths between 2 and 4 μm.

EXAMPLE 45

Insulinotropin (1 to 4 mg/mL) was dissolved in 1% sodium sulfate (orsodium acetate, or sodium chloride, or ammonium sulfate) solution athigher pH values than 8, and the pH of the solution was lowered down to6.0 to 7.5 with d-HCl. The clear solution was allowed to sit at ambienttemperature. After a couple of days, needle or plate shape crystals wereobtained depending on the crystallization conditions.

EXAMPLE 46

GLP-1 (7-37) was dissolved in 50 mM glycine buffer containing 0.1 to 0.2M NaCl at pH 8.5-9.5 at from 1 to 5 mg/ml. A solution of zinc salt(acetate or chloride) was added to obtain a molar ratio of from 0.5:1 to1.5:1 zinc:GLP-1 (7-37). Crystals of GLP-1 (7-37) grew overnight at roomtemperature with yields from 70 to 97%.

EXAMPLE 47

GLP-1 (7-37) crystals can be grown by vapor diffusion using the peptidedissolved in 100 mM Tris at pH 8-9.5 at from 10-20 mg/ml. The peptidesolution is mixed 1:1 with the same buffer containing from 0.5 to 2.5 MNaCl then equilibrated in a sealed system against the full strengthbuffer (i.e. Tris with 0.5-2.5 M NaCl).

1. A method for the treatment of non-insulin dependent diabetes mellitusin a mammal in need of such treatment comprising the repeatedadministration over an extended period of time of a compound withprolonged action after each administration, said prolonged actionnecessary to achieve sustained glycemic control in mammals.